stm.sciencemag.org/cgi/content/full/12/527/eaax3772/DC1

Supplementary Materials for

PI4KIIIβ is a therapeutic target in chromosome 1q–amplified lung adenocarcinoma

Xiaochao Tan, Priyam Banerjee, Edward A. Pham, Florentine U. N. Rutaganira, Kaustabh Basu, Neus Bota-Rabassedas,

Hou-Fu Guo, Caitlin L. Grzeskowiak, Xin Liu, Jiang Yu, Lei Shi, David H. Peng, B. Leticia Rodriguez, Jiaqi Zhang, Veronica Zheng, Dzifa Y. Duose, Luisa M. Solis, Barbara Mino, Maria Gabriela Raso, Carmen Behrens, Ignacio I. Wistuba,

Kenneth L. Scott, Mark Smith, Khanh Nguyen, Grace Lam, Ingrid Choong, Abhijit Mazumdar, Jamal L. Hill, Don L. Gibbons, Powel H. Brown, William K. Russell, Kevan Shokat, Chad J. Creighton*,

Jeffrey S. Glenn*, Jonathan M. Kurie*

*Corresponding author. Email: jkurie@mdanderson.org (J.M.K.); jeffrey.glenn@stanford.edu (J.S.G.);

creighto@bcm.edu (C.J.C.)

Published 22 January 2020, Sci. Transl. Med. 12, eaax3772 (2020) DOI: 10.1126/scitranslmed.aax3772

The PDF file includes:

Materials and Methods Fig. S1. Chromosome 1q is amplified in a subset of human lung cancer cell lines. Fig. S2. High expression of Golgi-related genes enhances the metastatic properties of 1q-amplified lung cancer cells. Fig. S3. 1q-amplified cancers are PI4KIIIβ dependent. Fig. S4. PI4KB functions cooperatively with coamplified genes on chromosome 1q. Fig. S5. IN-9 induces apoptosis in 1q-amplified, but not 1q-diploid, lung cancer cells. Fig. S6. Selective PI4KIIIβ antagonists have activity against 1q-amplified lung cancer cells. Fig. S7. PI4KIIIβ-dependent PI4P synthesis drives prometastatic properties of 1q-amplified lung cancer cells. Fig. S8. GOLPH3 mediates prometastatic effects of PI4KIIIβ in 1q-amplified cancer cells. Fig. S9. GOLPH3 mediates PI4KIIIβ-driven secretion. Fig. S10. 1q amplification is associated with increased secretion. Fig. S11. PLOD3 maintains H2122 cell survival by activating MMP9. Fig. S12. PI4KIIIβ-dependent secretion regulates processes in the tumor microenvironment. Table S1. Clinical pathological characteristics of lung adenocarcinomas. Table S2. Liquid chromatography–mass spectrometry of conditioned medium samples. Table S3. Primers. References (69–79)

Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/527/eaax3772/DC1)

Data file S1 (Microsoft Excel format). Original data.

Materials and Methods

Reagents

We purchased SYBR Green, fetal bovine serum (FBS), HEPES buffered medium, Dulbecco’s

minimal essential medium (DMEM), RPMI Medium 1640, Alexa Fluor-tagged secondary

antibodies, Cell-Light Golgi-GFP, and DAPI from Life Technologies; puromycin from

InvivoGene; paraformaldehyde from Electron Microscopy Sciences; Transwell and Matrigel-

coated Boyden chambers from BD Biosciences; G418 from Corning; PI(4)P mass ELISA kit

from Echelon (K-4000E); PI4KIIIβ inhibitor PI4KB-IN-9 (HY-19798) from MedChemExpress;

shRNAs against murine PI4KIIIβ (TRCN0000024759 and TRCN0000024763), human PI4KIIIβ

(TRCN0000199916 and TRCN0000199262), human PLOD3 (TRCN0000286656), human

SEMA3C (TRCN0000058132), human CLU (TRCN0000078611), human STC2

(TRCN0000331039) and TIMP1 (TRCN0000299344); siRNAs against murine PI4KIIIβ

(SASI_Mm01_00080645), human PI4KIIIβ (SASI_Hs01_00149544 and

SASI_Hs01_00149545), human GOLPH3L (SASI_Hs01_00163826 and

SASI_Hs01_00163830), human PI4KIIα (SASI_Hs01_00190417 and SASI_Hs01_00190418),

human RAB13 (SASI_Hs01_00081334 and SASI_Hs01_00081335), human VPS45

(SASI_Hs01_00165616 and SASI_Hs01_00165617), human GOLPH3 (SASI_Hs01_00163826

and SASI_Hs01_00163830), human FAPP1 (SASI_Hs01_00016597 and

SASI_Hs02_00352701), human CERT (SASI_Hs01_00081949 and SASI_Hs01_00081951),

human OSBP (SASI_Hs01_00068117 and SASI_Hs01_00068118), human PLOD3

(SASI_Hs01_00241346 and SASI_Hs02_00317421), human SEMA3C (SASI_Hs01_00161889

and SASI_Hs01_00161890), human CLU (SASI_Hs01_00066487 and SASI_Hs02_00332634),

human STC2 (SASI_Hs01_00025657 and SASI_Hs01_00025660), and human TIMP1

(SASI_Hs01_00019072 and SASI_Hs01_00019074) from Sigma. We purchased primary

antibodies against PI4KIIIβ (#611816) and GM130 (#560066) from BD Transduction

Laboratories; against PI4KIIIβ (NBP2-12814) from Novus Biologicals; against PI4P (Z-P004)

from Echelon Bioscience; against PRXD5 (17724-1-AP), ANXA2 (11256-1-AP), TIMP1 (16644-

1-AP), STC2 (10314-1-AP), SEMA3C (19242-1-AP), CLU (12289-1-AP) and PLOD3 (11027-1-

AP) from Proteintech; against α-tubulin (#T9026) from Sigma; against PARP-1 (#9542) and

cleaved-caspase 3 (#9664) from Cell Signaling; against VSV-G (IE9F9) from Kerafast; and

against TGN46 (AHP500G) from Bio-Rad. Antibodies used for flow cytometric detection of cells

in the tumor microenvironment and immunohistochemistry of tumor tissues are listed in

separate sections below. The EGFP-VSV-G (ts045) expression construct (Addgene plasmid

#11912) was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia). SAC1-K2A-gEGFP construct

was a gift from Peter Mayinger (Oregon Health & Science University). HA-PI4KB-pcDNA3.1 and

HA-PI4KB (D656A)-pcDNA3.1 were gifts from Tamas Balla (National Institutes of Health).

PI4KIIIβ inhibitor IN-9 (HY-19798) was purchased from MedChemExpress.

Cell lines

KP cells generated in mice that express K-rasG12D and p53R172H (393P, 344P and 344SQ) (33),

human lung cancer cells (A549, H1299, H460, H596, H23, H2122, HCC366, H1395, and

H3122), and the OVCAR-3 and TOV-21G human ovarian cancer cell lines were cultured in

RPMI 1640 containing 10% FBS. BEAS-2B immortalized human bronchial epithelial cells, 293T

human embryonic kidney cells, and human breast cancer cell lines (MCF-7, MDA-MB-468) were

cultured in DMEM containing 10% FBS. The CAOV-4 human ovarian cancer cell line was

cultured in L-15 Medium containing 20% FBS. Human umbilical vein endothelial cells (HUVECs)

were cultured in EGM-2 Endothelial Cell Growth Medium-2 BulletKit (CC-5035, Lonza). Cells

were maintained at 37°C in an incubator with a humidified atmosphere containing 5% CO2.

Cells were transfected with jetPRIME Versatile DNA/siRNA transfection reagent (Polyplus).

Stable cell transfectants were selected by using puromycin (for pLVX or pLKO.1 vectors) or

G418 (for pcDNA3.1 and pEGFP-C3 vectors).

Determination of compound A and B IC50 values against recombinant enzymes

IC50 values of compounds A and B against recombinant enzymes in solution were determined

as described (35).

Caco2 permeability assay

Caco-2 cells were maintained in DMEM in an atmosphere of 5% CO2. For transport experiments

50,000 cells/well were seeded on 12-well plates with polycarbonate filter inserts (Corning Costar

#3401) and allowed to grow and differentiate for 25 ± 4 days before the cell monolayers were

used for experiments. Apparent permeability coefficients were determined for apical to

basolateral and basolateral to apical directions. Test articles and reference compounds were

dissolved in Hank’s balanced salt solution (HBSS) containing 25 mM HEPES to yield a final

concentration of 10 μM. The assays were performed in HBSS at pH 7.4 for the basolateral side

and pH 6.5 for the apical side at 37 °C. Prior to the study, the monolayers were washed in

prewarmed HBSS. At the start of the experiments, prewarmed HBSS containing the test articles

was added to the donor side of the monolayer and HBSS without test articles was added to the

receiver side. Aliquots of the receiver side were taken over the 2-hour incubation period;

aliquots of the donor side were taken at 0 and 2 hours. Aliquots were diluted with an equal

volume of methanol/water with 0.1% formic acid containing the internal standard. The mixture

was analyzed by LC−MS/MS. The apparent permeability coefficients (Papp) were calculated

using the formula: Papp = (dCrec/dt)/(A × C0,donor)] × 106 with dCrec/dt being the change in

concentration in the receiver compartment with time; C0, donor the concentration in the donor

compartment at time 0; and A, the area of the cell monolayer.

Microsomal stability assay

Compounds (1 mM) were incubated in pooled human (BD#452117) and murine (BD#452701)

liver microsomes at 0.5 mg/mL final protein concentration. The reaction was started with

addition of 1 mM NADPH and stopped at 0, 10, 20, and 40 min with addition of cold acetonitrile

containing internal standard. Samples were analyzed by LC-MS/MS. Compounds’

disappearance rate constants were used to calculate half-life (t1/2).

Pharmacokinetic analysis of intravenously or orally administered compounds A and B

Compounds were formulated in 5% DMSO, 20% HPBCD, 10% PEG300, and 2% Poly80 and

either orally dosed at 10 mg/kg or intravenously dosed at 1 mg/kg to 8- to 10-week-old Balb/c

(Jackson Lab #651) mice. Serum was collected 0.5, 1, 2, 4, and 8 hours after a dose, and

analyzed by LCMS. Concentrations were imported into GraphPad Prism for area-under-the-

curve calculations (AUC) and half-life; AUCs were used to calculate bioavailability (F%).

Vector construction

The murine and human PI4KIIIβ coding sequences were isolated by performing PCR on cDNA

prepared from 344SQ and H1299 cells, respectively, and then cloned into pLVX-Puro and

pEGFP-C3 vectors (Clontech), respectively. Mouse PLOD3 coding sequence was isolated by

performing PCR on cDNA prepared from KC2 cells and cloned into pLVX-Blasticidin (modified

from pLVX-puro, Clonetech), and mutations were introduced by PCR method. PCR primers are

listed in table S3.

Cell proliferation, colony formation, apoptosis, migration, and invasion assays.

Cell proliferation assays were performed using Cell Proliferation Reagent WST-1 (Roche)

according to the manufacturer's protocol. For colony formation at low density on plastic, 500

cells were seeded per well into 6-well plates, and colonies were stained with 1% crystal violet

after 7-10 days. Colony formation assay in soft agar were performed as described previously

(69). For apoptosis detection by flow cytometry, tumor cells were isolated, suspended at 105/100

µl, incubated with Annexin V-FITC/propidium iodide solution using Dead Cell Apoptosis Kit

(ThermoFisher Scientific, V13242) according to manufacturer's instructions, and subjected to

flow cytometry to detect apoptotic cells. Migration and invasion assays were performed in

Transwell and Matrigel-coated Boyden chambers, respectively, as we have described (70).

Western blot analysis

For Western blot analysis, protein lysates were separated on a 10–12% Bis-Tris gel (Life

Technologies) and transferred to PVDF membranes. The membranes were blocked in 5% milk

and probed with primary antibodies following a standard protocol.

Lentivirus preparation

Lentiviral constructs that co-express Cre and PI4KIIIβ (lenti-CP) or GFP (lenti-CG) were

generated using the Gateway cloning system (Thermo Fisher Scientific). Lentivirus particles

were produced using standard lentivirus packaging vectors and preparation protocols,

concentrated by ultracentrifugation at 42,000 RPM for 30 min, and resuspended in Hank’s

Balanced Salt Solution. Lentivirus particle titers were determined by transducing a HEK293-Cre

reporter cell line as described previously (71).

qPCR analysis

Total RNA was isolated from cells using RNeasy Mini Kit (74106, Qiagen) and subjected to

reverse transcription using the qScript cDNA superMix (Quanta Biosciences). Genomic DNA

was isolated from cells using DNeasy Blood & Tissue Kits (69504, Qiagen). Gene copy

numbers and mRNA amounts were determined using SYBR Green Real-Time PCR Master

Mixes (Thermo Fisher Scientific) and normalized on the basis of ribosomal protein L32 (Rpl32)

mRNA. PCR primers are listed in table S3.

Digital droplet PCR

PI4KB (FAM) and RPP30 (HEX) probes (BioRad) were diluted in BioRad ddPCR supermix and

mixed with 4.4 units of HindIII restriction enzyme diluted in NEB buffer 2.1 (New England

BioLabs) to make a mastermix. 10 ng of each sample was added to the mastermix in a 96-well

plate. Each sample was run in duplicate. The droplets were automatically generated using the

Auto-DG (BioRad), after which they were amplified in a deep well thermocycler. The droplets

were detected using QX 200 droplet reader (BioRad) and analyzed with the Quantasoft

software. PI4KB copy number (normalized to RPP30) of each sample was determined based on

the ratio of normalized PCR values in tumor-to-normal lung.

ELISA

Intracellular PI4P amounts were determined using PI4P Mass ELISA Kit (K-4000E, Echelon

Biosciences) according to the manufacturer’s instructions. Briefly, acidic lipids were extracted

from whole cell lysates and the lipid extractions were added to the colored mixing plate with

PI4P grip. This mixture was then transferred to the PI4P detection plate for competitive binding.

A peroxidase-linked secondary detector and colorimetric detection were used to detect the

amount of PI4P grip binding to the plate. CLU and TIMP1 protein concentrations in conditioned

medium samples and cell lysates were determined using human Clusterin ELISA Kit (ab174447,

Abcam) and human TIMP1 ELISA Kit (ab187394, Abcam), respectively, according to the

manufacturer’s instructions.

Conditioned medium sample preparation and analysis

As previously described (72), conditioned medium samples were isolated, filtered through a

0.45-μm filter, mixed with an equal volume of complete growth medium (5% FBS final

concentration), applied to cells that had been seeded in 10 cm plates, and incubated in serum-

free medium for 16 hours. Conditioned medium in colony formation assays was replaced every

two days. Secreted factors in paired conditioned medium samples and cell lysates were

quantified by western blot analysis or ELISA. For both approaches, conditioned medium values

were normalized on the basis of cell lysate values using approaches described elsewhere (73-

75).

Cytokine and chemokine analysis

For each condition, 200,000 cells/well were plated in triplicate. Conditioned medium samples

were collected and frozen until analysis. Each sample was thawed, prepared, and analyzed

using a multiplex magnetic bead-based assay (Luminex 200 System, Luminex, and Multiplex

Analysis 5.1 software, Millipore-Sigma). Results represent mean values of triplicate samples.

Recombinant protein purification

Human PLOD3 recombinant protein was purified from Chinese hamster ovary cell–conditioned

medium as described previously (76). Briefly, PLOD3 recombinant protein (residues 32-738 of

LH3) containing N-terminal His8 and human growth hormone tags was produced as a secreted

protein with via large-scale transient transfection into Gibco ExpiCHO cells (Thermo Fisher

Scientific) with polyethylenimine. Conditioned medium samples were subjected to centrifugation

at 7000 rpm for 10 min, filtered through 0.22 µm EMD Millipore Stericup Sterile Vacuum Filter

Units (EMD Millipore), concentrated to 100 mL, buffer-exchanged into nickel-binding buffer (20

mM Tris, 200 mM NaCl, 15 mM imidazole, pH 8.0) using the Centramate & Centramate PE Lab

Tangential Flow System (Pall Life Sciences), and subjected to immobilized metal affinity

chromatography to purify recombinant PLOD3 protein.

MMP9 activity assay

H2122 cells were transfected with siRNAs against PLOD3 or TIMP1 or control siRNAs. After 48

h, medium was replaced with serum-free RPMI 1640, and conditioned medium samples were

collected 16 h later. MMP9 activity was quantified in conditioned medium samples using Human

Active MMP-9 Fluorokine E Kit (R&D Systems) following the manufacturer’s instructions.

Liquid chromatography-mass spectrometry of conditioned medium samples

Cells were seeded in 10-cm plates, and serum-free medium was added 24 h later. Conditioned

medium samples were collected 16 h later, filtered through 0.45 µm filter, and concentrated

consecutively using Amicon Ultra-15 10K and Ultra-0.5 10K centrifugal filters. Proteins were

separated by 1D gel electrophoresis, and Coomassie-stained bands were excised and

subjected to reduction using dithiothreitol and alkylation with iodoacetamide followed by tryptic

digestion. Briefly, the method consisted of a series of washing and dehydrating steps using 25

mM ABC (ammonium bicarbonate) and acetonitrile, respectively. The next step was reduction

via 10 mM DTT at 60°C for 30 min followed by alkylation with 50 mM iodoacetamide for 45 min

at room temperature (RT) in the dark. The gel spots then underwent another round of

washing/dehydrating steps prior to digestion with 100 ng trypsin at 37℃ overnight. Peptide

mixtures were analyzed by nanoflow liquid chromatography-tandem mass spectrometry

(nanoLC-MS/MS) using a nano-LC chromatography system (UltiMate 3000 RSLCnano,

Dionex), coupled on-line to a Thermo Orbitrap Fusion mass spectrometer (Thermo Fisher

Scientific) through a nanospray ion source (Thermo Scientific). A trap and elute method was

used. The trap column was a C18 PepMap100 (300 µm X 5 mm, 5 µm particle size)

(ThermoScientific). The analytical column used was Acclaim PepMap 100 (75 µm X 15 cm)

(Thermo Scientific). After equilibrating the column in 98% solvent A (0.1% formic acid in water)

and 2% solvent B (0.1% formic acid in acetonitrile), the samples (1 µL in solvent A) were

injected onto the trap column and subsequently eluted (400 nL/min) by gradient elution onto the

C18 column as follows: isocratic at 2% solvent B, 0-5 min; 2% to 45% solvent B, 2-37 min; 45%

to 90% solvent B, 37-40 min; isocratic at 90% solvent B, 40-45 min; 90% to 2%, 45-47 min; and

isocratic at 2% solvent B, 47-60 min. All LC-MS/MS data were acquired using XCalibur, version

2.1.0 (Thermo Fisher Scientific) in positive ion mode using a top speed data-dependent

acquisition method with a 3 sec cycle time. The survey scans (m/z 400-1600) were acquired in

the Orbitrap at 120,000 resolution (at m/z = 400) in profile mode, with a maximum injection time

of 50 msec and an automatic gain control (AGC) target of 200,000 ions. The S-lens RF level

was set to 60. Isolation was performed in the quadrupole with a 2.0 Da isolation window, and

higher-energy collisional dissociation MS/MS acquisition was performed in profile mode using

rapid scan rate with detection in the ion trap, with the following settings: parent threshold =

5,000; collision energy = 28%; maximum injection time 250 msec; AGC target 20,000 ions.

Monoisotopic precursor selection and charge state filtering were on, with charge states 2-6

included. Dynamic exclusion was used to remove selected precursor ions, with a +/- 10 ppm

mass tolerance, for 15 sec after acquisition of one MS/MS spectrum. Tandem mass spectra

were extracted and charge state deconvoluted by Proteome Discoverer (Thermo Fisher, version

1.4.1.14). All MS/MS spectra were searched against a Uniprot Human database using Sequest.

Searches were performed with a parent ion tolerance of 5 ppm and a fragment ion tolerance of

0.60 Da. Trypsin was specified as the enzyme, allowing for two missed cleavages. Fixed

modification of carbamidomethyl and variable modifications of oxidation and phosphorylation

were specified in Sequest.

Golgi-resident PI4P quantification

Cells were seeded on #1.5 cover glass-bottom wells and treated for 16 h with IN-9 or

transfected for 48 h with siRNAs against PI4KIIIβ. Cells were fixed with 3.7% methanol-free

paraformaldehyde for 20 min at RT, quenched with 50 mM NH4Cl for 15 min, and permeabilized

and blocked at 37℃ with staining buffer containing 0.2% saponin, 3% gelatin, 20 mM PIPES, pH

6.8, 137 mM NaCl, and 2.7 mM KCl. Primary antibodies against PI4P (1:250) and TGN46

(1:500) were added in staining buffer for 1 hour at 37℃ and incubated with AlexaFluor tagged

secondary antibodies (1:800) in staining buffer for 50 min at 37℃. Nuclei were counterstained

with DAPI, and cells were fixed post-staining with 2% paraformaldehyde for 5 min at RT. Cells

were imaged with confocal microscopy and analyzed by ImageJ. Masks were generated for the

thresholded area covered by TGN46 in each cell. Normalized Golgi PI4P amounts were

quantified by the ratio of the intensities of PI4P and TGN46 within the masked region.

VSV-G assay

Cells were transiently transfected with EGFP-VSV-G (ts045) plasmid. After 20-24 h, cells were

transferred to a restrictive temperature of 40°C for 18 h and then transferred to the permissive

temperature of 32°C in the presence of 100 mg/ml cycloheximide in RPMI supplemented with

0.2% FBS. Cells were fixed after 1 h, and exofacial VSV-G was detected in nonpermeabilized

cells by staining with IE9F9 (I14) anti-VSV-G monoclonal antibody. Total intracellular VSV-G

was detected from the EGFP signal. VSV-G trafficking to the plasma membrane was measured

by the ratio of exofacial (surface) VSV-G fluorescence signal to the EGFP (total) signal intensity

(n=15-20 cells per group).

Vesicular release assay

Cells were transiently transfected with EGFP-tagged STC2 expression vector. After 48 h, cells

were transferred to a restrictive temperature of 21.5°C for 2 hours in the presence of 100 μg/ml

cycloheximide in RPMI supplemented with 0.2% FBS and then transferred to the permissive

temperature of 37°C. Cells were fixed at pre-determined time points, permeabilized, and stained

with antibodies against a trans-Golgi network marker (TGN46) and a nuclear dye (DAPI). A

mask of the TGN46 channel was applied to the EGFP channel. Vesicular release was defined

as the reduction in EGFP signal intensity in TGN46-stained regions. The values were

normalized to T=0 (n=20 cells per group).

Microscopy and image analysis

Cells were imaged using an Eclipse Ti inverted microscope with A1+ confocal scanner (Nikon)

that has 405, 488, 561, and 640 nm wavelength diode lasers, high sensitivity Gallium arsenide

phosphide and photomultiplier tube detectors, and 60X 1.4 NA Oil or 100X 1.45 NA Oil

objectives. Images were acquired using NIS-Elements software (Nikon) version 4.40 (Build

1084). For high-resolution imaging, Z-stacks were acquired sequentially with slow scan speed,

512x512 or 1024x1024 frame size, low pinhole, and detector gain. Nyquist sampling criteria

were used along with optimal laser power to minimize bleaching. Post-acquisition, images were

processed and deconvolved with Huygens Professional version 18.04 (Scientific Volume

Imaging, The Netherlands, http://svi.nl) using the Classic Maximum Likelihood Estimation

algorithm. Images were analyzed using Fiji (ImageJ version 1.51s, NIH, http://imagej.nih.gov/ij,

Java 1.8.0_66, 64-bit), Huygens Professional, or NIS-Elements. Immunofluorescence

procedures were performed as described previously (70). For fluorescence intensity

measurements in VSV-G and vesicular release assays, Z-stacks were projected using ‘sum of

the slices’ algorithm, and normalized mean fluorescence intensity (NMF) was measured by the

following equation: NMF = ID – (A X Bg), where ID is the integrated pixel density within selected

ROI, A is the area of the ROI, and Bg is the mean background fluorescence intensity.

Immunohistochemical analysis of tumor tissues

Using an automated immunostainer platform, 4 μm tissue sections from formalin-fixed and

paraffin-embedded lung tissues were stained in a Leica Bond Max automated stainer (Leica

Biosystems Nussloch GmbH). The tissue sections were deparaffinized and rehydrated following

the Leica Bond protocol. Antigen retrieval was performed with Bond Solution #2 (Leica

Biosystems, equivalent EDTA, pH 9.0) for 30 min; the primary antibodies (PI4KIIIβ, dilution 1:25,

Novus Biological, NBP1-80906; CD31, rabbit monoclonal antibody clone D8V9E, dilution 1:100,

Cell Signaling Technology, #77699; αSMA, dilution 1:300, Abcam, ab5694) were incubated for

15 min at RT. The primary antibody was detected using the Bond Polymer Refine Detection kit

(Leica Biosystems) with DAB as chromogen. The slides were counterstained with hematoxylin,

dehydrated, and cover slipped. The immunostained sections were digitally scanned using the

Aperio AT2 slide scanner (Leica Biosystems) under 20 × objective magnification. We used

digital image analysis with pathologist-trained specific algorithms to quantify: a) the extent and

intensity of PI4KIIIβ expression in malignant cells and calculate an H-score (0-300)

(“Cytoplasmic v2”, Aperio Bright field Toolbox image analysis software, Leica Biosystems). b)

CD31-positive micro vessel density (“Micro vessel analysis v1”, Aperio Bright field Toolbox

image analysis software, Leica Biosystems), and c) α-smooth muscle actin percentage of

positive tumor area (“Area Quantification” algorithm, HALO image analysis software, Indica

Labs). Bright field algorithms use color deconvolution to separate chromogenic stains for

analysis. H-scores were calculated as we have described (77).

Multicellular aggregates containing CAFs and cancer cells

Primary CAFs were isolated from tumor-bearing lungs of KrasLA1 mice as described previously

(78) and stably transfected with green fluorescence protein expression vector. Multicellular

aggregates containing 344SQ cells alone (n=50 cells) or 344SQ cells and CAFs (50:30 ratio)

were generated by seeding the cells in laser-ablated microwells (79), allowed to aggregate for

48 h, and embedded in rat tail collagen I (Serva, 47256.01) gel at a final concentration of 2

mg/ml (200 µl/gel, 35 aggregates/gel). The gel was left to polymerize on a glass-bottom 35 mm

dish (Mattek), at 370 C for 30 min. The aggregates were allowed to invade for 2 days in the cell

culture incubator (370 C, 5% CO2), followed by fixation with 4% PFA for 20 min at RT. After

fixation, gels were stained with phalloidin (AlexaFluor-568) to visualize the tumor cells. Images

of PFA-fixed aggregates in 3D collagen gels were acquired with a NikonA1 confocal

microscope, 10x objective. After collecting z-slices, the structure volume was rendered into 2D

projections by maximum intensity projection algorithm (ImageJ), and invasive structures were

analyzed. An invasive projection was defined as having at least one cell protruding out of the

aggregate. A leader follower cell structure was defined as an invasive projection containing at

least one CAF at the tip and collectively invasive tumor cells that follow behind. Invasive tumor

cell projections with or without CAFs at the tip were manually counted.

Tube formation assay

H2122 cells (1×105/well) and human umbilical vein endothelial cells (HUVECs) (1×105/well)

were seeded into the upper and lower wells, respectively, of Transwell plates. Lower wells were

Matrigel-coated. After co-culture for 8 h in RPMI1640 medium supplemented with 1% FBS,

HUVEC cells were photographed under a phase-contrast microscope (10×) and tube-like

structures were quantified from 10 randomly chosen fields.

Immune cell quantification in tumor tissues

Three weeks after subcutaneous injection of 1×106 344SQ_shCTL or 344SQ_shPI4KIIIβ into

the flank of wild-type mice, subcutaneous tumors were processed using the MACS Miltenyl

Biotec tumor dissociation kit. Digestion was performed using collagenase I (3 mg/ml) and

dispase II (4 mg/ml). Spleens were processed by grinding tissues using a 40 micron nylon

filter. After single cell suspensions were obtained, RBCs were lysed using 1 x RBC lysis buffer

(Biolegend) following manufacturer instructions. Single cell suspension was stained according to

standard protocols with the following antibodies: CD3-PE-594 (BioLegend, 100246), CD45-

Pacific Blue (BioLegend, 103126), CD4-APCCy7 (BioLegend, 100526), CD8-PE-Cy7 (100721),

CD278-PE (BioLegend, 117406), CD25-BUV395 (BD Biosciences, 564022), PD1-BV-505

(BioLegend,135220), CD62L-FITC (FisherScientific, 35-0621-U500), CD44-BV-711 (BioLegend,

103057), TIM3-APC (BioLegend, 134007),FoxP3-PerCP-Cy5.5 (Invitrogen, 45-5773-82), GR1-

BV-711 (BioLegend, 106443), CD11b-BV-650 (BioLegend, 101239), CD11c-BV-785

(BioLegend, 117335), F4/80-APC (Tonbo, 204801-U100), PDL1-PE-Dazzle-594 (BioLegend,

124323), CD86-APC-Cy7 (Biolegend, 105030),MCHII-PE-Cy7 (107629), CD80-BV-605

(BioLegend, 104729), CD68-PerCP-Cy5.5 (BioLegend,137009), iNOS-PE (Invitrogen, 125920-

80), Arg1-FITC (RDSystems, IC5868F), CD31-BV-786 (BD Biosciences 740870), and the

live/dead cell marker Ghost Violet-BV-510 (VWR 10-0870-T100). For intracellular staining, cells

were fixed and permeabilized using the intracellular staining perm wash buffer (BioLegend)

according to manufacturer instructions. Data were acquired on a Fortessa X20 analyzer (BD

Biosciences) and analyzed using FlowJo software (version 7.6; Tree Star).

Myeloid-derived suppressor cell quantification in splenocyte/cancer cell co-cultures

Spleens from wild-type 129/sv female mice were harvested, dissociated, and passed through 40

µm Falcon Nylon Single Cell Strainers (Fisher Scientific). Spleens were centrifuged at 2000

RPM for 5 min and resuspended in 1x RBC Lysis Buffer (Biolegend) for 2 min at RT. RPMI 1640

was added to stop the lysis procedure and spleens were centrifuged as before and

resuspended in fresh RPMI 1640 + 10% FBS. For co-culture experiments, cancer cell lines

were seeded in 24-well tissue culture plates and isolated splenocytes were added to the cells at

a 2:1 splenocyte-to-cancer cell ratio with 1 µg/mL of anti-mouse CD3 and CD28 antibody

(Biolegend). Splenocytes were co-cultured with cancer cells for 48 and 96 hours, after which

splenocytes were collected from the culture medium, centrifuged, and fixed with 1%

paraformaldehyde for 30 min at RT. MDSC populations were analyzed by FACS using

CD45+CD11b+GR-1+ gated cells from total splenocyte populations.

Supplementary Figures

Fig. S1. Chromosome 1q is amplified in a subset of human lung cancer cell lines. (A)

Copy numbers of 1q21.3-encoded genes in human lung cancer cell lines with or without 1q

amplifications. Immortalized bronchial epithelial (BEAS-2B) cells included as control. (B)

Western blot analysis (gels) of PI4KIIIβ protein amounts in 1q-amplified (red) and 1q-diploid

(black) lung cancer cell lines quantified by densitometry (bar graph).

A B

PI4KIIIβ

α-Tubulin

BE

AS

-2B

A549

H1299

H460

H23

HC

C36

6

H21

22

H1395

H3122

1.0 1.1 1.6 1.5 2.4 2.5 2.5 3.2 3.5

1q-diploid 1q-amplified

Amp.1q21.3 copy number > 3

Gene c

opy n

um

ber

BE

AS

-2B

A5

49

H1

29

9

H4

60

H5

96

H2

12

2

H2

3

HC

C3

66

H1

39

5

H3

12

2

0

4

8

12

16

GOLPH3LRAB13VPS45

PI4KB

Rela

tive P

I4K

III

pro

tein

expre

ssio

n

BE

AS

-2B

A5

49

H1

29

9H

46

0H

23

HC

C3

66

H2

12

2H

13

95

H3

12

2

0

1

2

3

4 P=0.001

Fig. S2. High expression of Golgi-related genes enhances the metastatic properties of 1q-

amplified lung cancer cells. (A) Quantitative RT-PCR (qPCR) analysis of mRNA expression in

H2122 cells transfected with the indicated small interfering RNAs (siRNAs). (B) Colonies formed

on plastic (adherent) and in soft agar (non-adherent) by H2122 cells transfected with the

H 2 1 2 2

Ce

ll p

roli

fera

tio

n (

OD

45

0)

D a y 1 D a y 2 D a y 3 D a y 4

0 .0

0 .3

0 .6

0 .9

1 .2s iC T L

s iP I4 K III

s iG O L P H 3 L

s iR A B 1 3

P = 0 .0 0 4

s iV P S 4 5

siCTL siPI4KIIIβ siGOLPH3L siRAB13 siVPS45

H2122A B

J

E F G

H 2 3

Re

lati

ve

ce

ll n

um

be

r

pe

r fi

eld

m ig ra t io n in v a s io n

0 .0

0 .5

1 .0

1 .5 s iC T L

s iP I4 K III

s iG O L P H 3 LP < 0 .0 0 1

s iR A B 1 3

s iV P S 4 5

P < 0 .0 0 1

P = 0 .0 1

P = 0 .0 0 7P = 0 .0 2

adherent

non-

adherent

H 2 3

Ce

ll p

roli

fera

tio

n (

OD

45

0)

D a y 1 D a y 2 D a y 3 D a y 4

0 .0

0 .5

1 .0

1 .5

2 .0 s iC T L

s iP I4 K III

s iG O L P H 3 L

s iR A B 1 3

P < 0 .0 0 1

s iV P S 4 5

C D

H 3 1 2 2

Re

lati

ve

co

lon

y n

um

be

r

siC

TL

siP

I4K

III

#1

siP

I4K

III

#2

0 .0

0 .5

1 .0

1 .5

P < 0 .0 0 1

P < 0 .0 0 1

H 2 3

Re

lati

ve

co

lon

y n

um

be

r

shC

TL

siP

I4K

III

#1

siP

I4K

III

#2

0 .0

0 .5

1 .0

1 .5

P = 0 .0 1

P = 0 .0 0 8

H I

H3122

PI4KIIIβ

α-Tubulin

siCTL siPI4KIIIβ#1 siPI4KIIIβ#2

H3122

H23

siCTL siPI4KIIIβ siGOLPH3L siRAB13 siVPS45

invasio

n

mig

ration

H23

PI4KB GOLPH3L RAB13 VPS450.0

0.5

1.0

1.5

H2122

Rela

tive m

RN

A e

xpre

ssio

n

siCTLsiPI4KIIIsiGOLPH3LsiRAB13siVPS45

*** *** *** ***

*** P<0.001

H 2 1 2 2

Re

lati

ve

co

lon

y n

um

be

r

0 .0

0 .5

1 .0

1 .5

P < 0 .0 0 1

P = 0 .0 0 6

P < 0 .0 0 1

adherent Non-adherent

H2122

Rela

tive c

olo

ny n

um

ber

0.0

0.5

1.0

1.5P<0.001P=0.04

P<0.001 siCTL

siPI4KIIIsiGOLPH3L

siVPS45siRAB13

H3122C

ell

pro

lifera

tion (

OD

450)

Day 1 Day 2 Day 3 Day 40.0

0.5

1.0

1.5

2.0

2.5siCTLsiPI4KIII#1siPI4KIII#2

***

***

***P<0.001

PI4KB GOLPH3L RAB13 VPS450.0

0.5

1.0

1.5

2.0

H23

Rela

tive m

RN

A e

xpre

ssio

n

siCTLsiPI4KIIIsiGOLPH3LsiRAB13siVPS45

****** ***

***

*** P<0.001

indicated siRNAs. (C) Quantification of colony numbers in (B). Values expressed relative to

siCTL-transfected cells, which were set at 1.0. (D) Proliferation of cells grown on plastic

determined by WST-1 assays. (E) qPCR analysis of mRNA expression in H2122 cells

transfected with the indicated siRNAs. (F) Cell proliferation determined by WST-1 assays. (G)

Western blot analysis of H3122 cells transfected with indicated siRNAs. (H) Colonies formed in

soft agar. (I) Relative cell densities determined by WST-1 assays. (J) Migrated and invaded

cells in Transwell chambers. Scale bars: 200 μm.

Fig. S3. 1q-amplified cancers are PI4KIIIβ dependent. (A and B) PI4KB gene copy-numbers

(A) and mRNA expression (B) in 1q-amplified breast cancer (MCF-7 and MDA-MB-468) and

ovarian cancer (OVCAR-3 and CAOV-4) cell lines. Values expressed relative to 1q-diploid cell

lines (MCF10A and TOV21G). (C) Western blot analysis of 1q-amplified cell lines transfected

with siCTL or siPI4KIIIβ. (D) Relative cell densities determined by WST-1 assays. (E) Colonies

formed on plastic by MCF-7 breast cancer cells. (F) Migrated and invaded MDA-MB-468 cells in

Transwell chambers. Scale bar: 200 μm.

MC

F10A

MC

F-7

MD

A-M

B-4

68

TO

V21G

OV

CA

R-3

CA

OV

-4

0

1

2

3

4

5

PI4

KB

ge

ne

co

py

nu

mb

er

M C F -7

Ce

ll p

roli

fera

tio

n (

OD

45

0)

D a y 1 D a y 2 D a y 3 D a y 4

0 .0

0 .5

1 .0

1 .5

2 .0s iC T L

s iP I4 K III # 1

s iP I4 K III # 2***

***

***P < 0 .0 0 1

A B

E

C

D

M C F -7

Re

lati

ve

co

lon

y n

um

be

r

0 .0

0 .5

1 .0

1 .5P = 0 .0 0 8

P < 0 .0 0 1s iC T L

s iP I4 K III # 1

s iP I4 K III # 2

OVCAR-3 CAOV-4 MCF-7 MDA-MB-468

PI4KIIIβ

α-Tubulin

M B -M D A -4 6 8

Re

lati

ve

ce

ll n

um

be

r

pe

r fi

eld

m ig ra t io n in v a s io n

0 .0

0 .5

1 .0

1 .5 s iC T L

s iP I4 K III # 1

s iP I4 K III # 2

P = 0 .0 0 1

P < 0 .0 0 1 P < 0 .0 0 1

P = 0 .0 0 1

M B -M D A -4 6 8

Re

lati

ve

ce

ll n

um

be

r

pe

r fi

eld

m ig ra t io n in v a s io n

0 .0

0 .5

1 .0

1 .5 s iC T L

s iP I4 K III # 1

s iP I4 K III # 2

P = 0 .0 0 1

P < 0 .0 0 1 P < 0 .0 0 1

P = 0 .0 0 1

siCTL siPI4KIIIβ#1 siPI4KIIIβ#2

F

MCF-7

siCTL siPI4KIIIβ#1 siPI4KIIIβ#2

Invasio

n

mig

ration

MB-MDA-468

MCF10

A

MCF-7

MDA-M

B-4

68

TOV21

G

OVCAR-3

CAOV-4

2 -1

20

21

22

23

24

25

Rela

tive e

xpre

ssio

n o

f

PI4

KB

mR

NA

MB-MDA-468

Cell

pro

lifera

tion (

OD

450)

Day 1 Day 2 Day 3 Day 40.0

0.3

0.6

0.9

1.2siCTLsiPI4KIII#1siPI4KIII#2

***

***P<0.001

***

OVCAR-3

Cell

pro

lifera

tion (

OD

450)

Day 1 Day 2 Day 3 Day 40.0

0.3

0.6

0.9

1.2siCTLsiPI4KIII#1siPI4KIII#2

******

***P<0.001

CAOV-4

Cell

pro

lifera

tion (

OD

450)

Day 1 Day 2 Day 3 Day 40.0

0.3

0.6

0.9

1.2siCTLsiPI4KIII#1siPI4KIII#2

*********P<0.001

Fig. S4. PI4KB functions cooperatively with coamplified genes on chromosome 1q. (A)

qPCR analysis of ectopically expressed genes in H1299 cells. Values normalized on the basis

of RPL32 expression and expressed relative to GFP-transfected cells. (B and C) Invaded cells

in Transwell chambers were photographed (B) and quantified (C). Scale bar: 200 μm. (D)

Western blot analysis of ectopically expressed proteins in 344P cells. Empty vector (mCherry).

(E) 344P flank tumor weights (left) and lung metastasis numbers (right) per mouse.

A B

GF

P

GO

LP

H3

L

VP

S4

5

GP

R8

9A

RA

B1

3

SC

AM

P3

TM

EM

79

VA

NG

L2

BL

ZF

1

KL

HL

20

VA

MP

4

KL

HL

12

GO

LT

A1

ST

X6

RF

WD

2

QS

OX

1

RA

B2

9

AC

BD

3

AR

F1

NM

NA

T2

0

1

2

3

4

5

Re

lati

ve

in

va

sio

n

**

*****

*

**

*

***

H 1 2 9 9

H1299_PI4KIIIβ

GPR89A

ACBD3

GFP-PI4KIIIβPI4KIIIβ

α-Tubulin

mC

he

rry

mC

he

rry

AC

BD

3

GP

R8

9A

Vec PI4KIIIβ

344P E

Me

tas

tas

is (

n)

0

2

4

6

8

1 0P = 0 .0 4

V e c + m C h e rry

P I4 K III + m C h e rry

P I4 K III + A C B D 3

P I4 K III + G P R 8 9 AP = 0 .0 4

Tu

mo

r s

ize

(g

)

0 .0

0 .4

0 .8

1 .2

1 .6

P < 0 .0 0 1

P < 0 .0 0 1

P < 0 .0 5

Me

tas

tas

is (

n)

0

2

4

6

8

1 0P = 0 .0 4

V e c + m C h e rry

P I4 K III + m C h e rry

P I4 K III + A C B D 3

P I4 K III + G P R 8 9 AP = 0 .0 4

344P

D

C

GFP GOLPH3L VPS45 GPR89A RAB13

SCAMP3 TMEM79 VANGL2 BLZF1 KLHL20

VAMP4 KLHL12 GOLTA1 STX6 RFWD2

QSOX1 RAB29 ACBD3 ARF1 NMNAT2

Invasion

H1299_PI4KIIIβ

GO

LP

H3L

VP

S4

5G

PR

89A

RA

B1

3S

CA

MP

3T

ME

M7

9V

AN

GL2

BL

ZF

1K

LH

L2

0V

AM

P4

KL

HL

12

GO

LT

A1

ST

X6

RF

WD

2Q

SO

X1

RA

B2

9A

CB

D3

AR

F1

NM

NA

T2

100

101

102

103

104F

old

change o

f in

dic

ate

d

co-e

xpre

ssed g

enes (

/GF

P)

H1299_PI4KIIIβ

Fig. S5. IN-9 induces apoptosis in 1q-amplified, but not 1q-diploid, lung cancer cells.

Western blot analysis to detect cleaved PARP and cleaved caspase 3 in cells treated with IN-9.

α-tubulin included as loading control.

PARP1Cleaved PARP1

Cleaved Caspase3

α-Tubulin

H2122 H23 H3122 A549 H460 H1299

IN-9 (µM): 0 5 10 0 5 10 0 5 10 0 5 10

1q-amplified 1q-diploid

0 5 10 0 5 10

Fig. S6. Selective PI4KIIIβ antagonists have activity against 1q-amplified lung cancer

cells. (A, B) Chemical structures and IC50 values of compounds A (A) and B (B). IC50 values

determined in cell-free assays using indicated recombinant human enzymes. Cell permeability

determined in Caco-2 permeability assays. Apparent permeability coefficients (Papp, 1 x 10-6

cm/sec): A to B, apical to basolateral; B to A, basolateral to apical; BA/AB ratio. Metabolic

Re

lati

ve

co

lon

y n

um

be

r

H 3 1 2 2 H 2 1 2 2 H 2 3 H 4 6 0 H 1 2 9 9 A 5 4 9

0 .0

0 .5

1 .0

1 .5

0 M

1 M

C o m p o u n d B

**

***

*P < 0 .0 5

**P < 0 .0 1

***P < 0 .0 0 1

1 q -a m p lif ie d 1 q -d ip lo id

**

**

*

*

A

C

Compound B (µM): 0 1 0 1

1q-amplified

H3122

H2122

H23 A549

H460

H1299

1q-diploid

B

D

H2122

A (μM): 0 0.5 1 2

adherent

Non-

adherentR

ela

tiv

e c

olo

ny

nu

mb

er

0 0 .5 1 2

0 .0

0 .5

1 .0

1 .5

C o m p o u n d A (M )

****** ***

*** P < 0 .0 0 1

Re

lati

ve

co

lon

y n

um

be

r

0 0 .5 1 2

0 .0

0 .5

1 .0

1 .5

C o m p o u n d A (M )

***

******

*** P < 0 .0 0 1

adherent

non-adherent

DMSO Compound B

Invasio

n

m

igra

tion

H23H 2 3

Re

lati

ve

ce

ll n

um

be

r

m ig ra t io n in v a s io n

0 .0

0 .5

1 .0

1 .5D M S O

C o m p o u n d B

P = 0 .0 0 2P < 0 .0 0 1

H 2 3

Re

lati

ve

ce

ll n

um

be

r

m ig ra t io n in v a s io n

0 .0

0 .5

1 .0

1 .5D M S O

C o m p o u n d A

P = 0 .0 1 7 P = 0 .0 0 1

E F G

0 2 4 6 8 1 0

5 0

7 5

1 0 0

1 2 5

1 5 0

D a y a fte r tre a tm e n tMo

us

e b

od

y w

eig

ht

ch

an

ge

(%

)

V e h ic le

C o m p o u n d A

Body w

eig

ht

Change (

%)

0 5 1 0 1 5 2 0

5 0

7 5

1 0 0

1 2 5

1 5 0

D a y a fte r tre a tm e n tMo

us

e b

od

y w

eig

ht

ch

an

ge

(%

)

V e h ic le

C o m p o u n d B (2 0 m g /k g )

C o m p o u n d B (4 0 m g /k g )

Body w

eig

ht

Change (

%)

H I J

Compound API4KIIIa IC50 = 36 nM

PI4KIIIb IC50 = 20 nM

PI3Kb IC50 = >50 µM

PI3Kγ IC50 = >50 µM

PI3KC2γ IC50 = 6.2 µM

Caco2 Papp x 1e-6 cm/s A to B 2.4

Caco2 Papp x 1e-6 cm/s B to A 400

Caco2 Papp x 1e-6 cm/s BA/AB ratio 167

Metabolic stability HLM/MLM (t1/2 min) 72 /33

Mouse PK 10 mg/Kg PO F% 2.6

Compound BPI4KIIIa IC50 = 11 µM

PI4KIIIb IC50 = 23 nM

PI3Kb IC50 = >50 µM

PI3Kγ IC50 = >50 µM

PI3KC2γ IC50 = >50 µM

Caco2 Papp x 1e-6 cm/s A to B 10

Caco2 Papp x 1e-6 cm/s B to A 18

Caco2 Papp x 1e-6 cm/s BA/AB ratio 1.8

Metabolic stability HLM/MLM (t1/2 min) 40 /9

Mouse PK 10 mg/Kg PO F% 100

Mouse PK 10 mg/Kg PO Cmax 1.07 µM

T1/2 (h) = 1

stability in murine (MLM) and human (HLM) liver microsomes. Mouse pharmacokinetic (PK)

parameters (T1/2, Cmax, F%) determined at 10 mg/kg dose. (C) Colonies formed in soft agar

quantified after 7 days of compound B treatment. (D) Quantification of colony numbers in (C).

(E) Migrated and invaded H23 cells in Transwell chambers quantified after 10 h of treatment. (F

and G) Colonies formed on plastic (adherent) and in soft agar (non-adherent) were imaged (F)

and quantified (G) after 7 d of compound A treatment. (H) Migrated and invaded H23 cells in

Transwell chambers quantified after 10 h of compound A treatment. (I and J) Daily weights of

mice treated with vehicle or compound A (I) or compound B (J). Scale bars: 200 μm.

Fig. S7. PI4KIIIβ-dependent PI4P synthesis drives prometastatic properties of 1q-

amplified lung cancer cells. (A) Golgi localization of EGFP-tagged SAC1-K2A was determined

by co-localization with Golgi marker GM130. Scale bar: 20 μm. (B) Western blot confirmation of

ectopic SAC1-K2A expression in H1299 and A549 cells. (C and D) Relative cell densities

α-Tubulin

EGFP

SAC1-K2A

A549 H1299

A 5 4 9

Ce

ll p

roli

fera

tio

n (

OD

45

0)

D a y 1 D a y 2 D a y 3 D a y 4

0 .0

0 .5

1 .0

1 .5

2 .0V e c

S A C 1 -K 2 A

H 1 2 9 9

Ce

ll p

roli

fera

tio

n (

OD

45

0)

D a y 1 D a y 2 D a y 3 D a y 4

0 .0

0 .2

0 .4

0 .6

0 .8V e c

S A C 1 -K 2 A

A549

H1299

Re

lati

ve

co

lon

y n

um

be

r

A 5 4 9 H 1 2 9 9

0 .0

0 .5

1 .0

1 .5E G F P

S A C 1 -K 2 AP = 0 .0 2 1

EGFP SAC1-K2A

Re

lati

ve

ce

ll n

um

be

r

m ig ra t io n in v a s io n m ig ra t io n in v a s io n

0 .0

0 .5

1 .0

1 .5E G F P

S A C 1 -K 2 A

P = 0 .0 2 6 P = 0 .0 0 2

A 5 4 9 H 1 2 9 9

P = 0 .0 1 1P = 0 .0 2 2

Inva

sio

n

mig

ratio

nEGFP SAC1-K2AEGFP SAC1-K2A

A549 H1299

siCTL siPI4KIIα#1 siPI4KIIα#2

inva

sio

n

m

igra

tio

n

SAC1-K2A GM130 Merge

A B C

D E

F G

I

H

J

K

% A

po

pto

tic

ce

lls

siC

TL

siP

I4K

II

siP

I4K

III

0

5

1 0

1 5

P = 0 .0 2

H 2 1 2 2

H 2 3

Ce

ll p

roli

fera

tio

n (

OD

45

0)

D a y 1 D a y 2 D a y 3 D a y 4

0

1

2

3

s iC T L

s iP I4 K II # 1

s iP I4 K II # 2

*** P < 0 .0 0 1 ***

***

H 2 3

Re

lati

ve

ce

ll n

um

be

r

m ig ra t io n in v a s io n

0 .0

0 .5

1 .0

1 .5

2 .0 s iC T L

s iP I4 K II # 1P < 0 .0 0 1 s iP I4 K II # 2P < 0 .0 0 1

P = 0 .0 1

P = 0 .0 2

L

H23

Rela

tive e

xpre

ssio

n

ofP

I4K

2A

mR

NA

siCTL

siPI4

KII

#1

siPI4

KII

#20.0

0.5

1.0

1.5

P<0.001

P<0.001

0 2 5 10 0 2 5 100

5

10

15

20

25

30

PI4

P c

oncentr

ation

(pm

ol/10

6 c

ells

)

IN-9 (M)

H1299_Vec H1299_PI4KIII

P=0.01P=0.005

P=0.007P=0.001

H2122

Rela

tive m

RN

A e

xpre

ssio

n

PI4K2A PI4KB0.0

0.5

1.0

1.5

P<0.001

siCTL

siPI4KII

siPI4KIII

P<0.001

determined by WST-1 assays in A549 (C) and H1299 (D) cells. (E) Invaded cells in Transwell

chambers were quantified. Scale bars: 200 μm. (F) Colonies formed in soft agar were

quantified. (G) Total cellular PI4P concentrations in H1299_vector cells and H1299_PI4KIIIβ

cells determined by ELISA. (H) qPCR analysis of PI4K2A mRNA expression in siRNA-

transfected cells. (I) Relative cell densities determined by WST-1 assays. (J) Migrated and

invaded cells in Transwell chambers. Scale bar: 200 μm. (K) qPCR analysis of PI4K2A and

PI4KB mRNA expression in siRNA-transfected cells. (L) Apoptotic cells detected by flow

cytometry. Values expressed as % of total cells analyzed.

Fig. S8. GOLPH3 mediates prometastatic effects of PI4KIIIβ in 1q-amplified cancer cells.

(A) Genomic alterations (rows) in TCGA lung adenocarcinomas (columns). (B) Western blot

analysis of H23 cells and H2122 cells transfected with siCTL or siGOLPH3. (C) Relative cell

densities determined by WST-1 assays. (D) Colonies formed on plastic (adherent) and in soft

H 2 1 2 2

Ce

ll p

roli

fera

tio

n (

OD

45

0)

D a y 1 D a y 2 D a y 3 D a y 4

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

3 .0 s iC T L

s iO S B P

s iF A P P 1

s iC E R T

H 2 1 2 2

Ce

ll p

roli

fera

tio

n (

OD

45

0)

D a y 1 D a y 2 D a y 3 D a y 4

0 .0

0 .3

0 .6

0 .9

1 .2

1 .5s iN C

s iG O L P H 3 # 1

s iG O L P H 3 # 2

******

*** P < 0 .0 0 1

A B C

D E F

GOLPH3

α-Tubulin

H23 H2122

H 2 3

Re

lati

ve

ce

ll n

um

be

r

pe

r fi

eld

m ig ra t io n in v a s io n

0 .0

0 .5

1 .0

1 .5

2 .0 s iC T L

s iG O L P H 3 # 1

s iG O L P H 3 # 2P = 0 .0 0 5

P = 0 .0 0 6P = 0 .0 0 1

P = 0 .0 0 2

G H I

J K

H 1 2 9 9

Ce

ll p

roli

fera

tio

n (

OD

45

0)

D a y 1 D a y 2 D a y 3 D a y 4

0 .0

0 .5

1 .0

1 .5 V e c + s iC T L

P I4 K III + s iC T L

P I4 K III +

s iG O L P H 3

***P < 0 .0 0 1

******

L M

H 2 1 2 2

Re

lati

ve

co

lon

y n

um

be

r

0 .0

0 .5

1 .0

1 .5

P < 0 .0 0 1

P < 0 .0 0 1

H 2 1 2 2

Re

lati

ve

co

lon

y n

um

be

r

0 .0

0 .5

1 .0

1 .5

P < 0 .0 0 1

P < 0 .0 0 1s iC T L

s iG O L P H 3 # 1

s iG O L P H 3 # 2

CFA SACFA

GOLPH3

β-actin

PI4KIIIβ

Vec

PI4KIIIβ

siCTL

siGOLPH3

+ - -

- + +

+ + -

- - +

H1299

adherent non-adherent

siCTL siGOLPH3#1 siGOLPH3#2

invasio

n

mig

ratio

n

H23

siCTL siOSBP siFAPP1 siCERT

H2122

siCTL siGOLPH3#1 siGOLPH3#2

adherent

non-adherent

H2122

adherent

non-adherent

adherent non-adherent

H 2 1 2 2

Re

lati

ve

co

lon

y n

um

be

r

0 .0

0 .5

1 .0

1 .5s iC T L

s iO S B P

s iF A P P 1

s iC E R T

siCTL siCTL siGOLPH3

PI4KIIIβVec

Re

lati

ve

ce

ll n

um

be

r

pe

r fi

eld

0

1

2

3 V e c + s iC T L

P I4 K III + s iC T L

P I4 K III + s iG O L P H 3

P < 0 .0 0 1

H 1 2 9 9

P < 0 .0 0 1H1299

invasionOSBP FAPP1 CERT

0.0

0.5

1.0

1.5

2.0

H2122

Rela

tive e

xpre

ssio

n

of

mR

NA

s

siCTLsiOSBPsiFAPP1siCERT

*** *** ***

*** P<0.001

Rela

tive c

olo

ny n

um

ber

0.0

0.5

1.0

1.5

Rela

tive c

olo

ny n

um

ber

0.0

0.5

1.0

1.5siCTLsiOSBPsiFAPP1siCERT

agar (non-adherent). (E) Quantification of colonies in (D). (F) Migrated and invaded cells in

Transwell chambers. (G) Quantification of cells in (F). (H) Western blot analysis of ectopic

PI4KIIIβ expression and GOLPH3 depletion in H1299 cells. (I) Relative cell densities determined

by WST-1 assays. (J) Invaded cells in Transwell chambers. (K) qPCR analysis of mRNA

expression in H23 cells transfected with the indicated siRNAs. (L) Relative cell densities

determined by WST-1 assays. (M) Colonies formed on plastic (adherent) and in soft agar (non-

adherent). Scale bars: 200 μm.

Fig. S9. GOLPH3 mediates PI4KIIIβ-driven secretion. (A) Quantification of protein bands in

Fig. 6I using ImageJ (n=3 per condition). (B and C) Western blot analysis of TIMP1 and CLU

protein expression in CM (B) and cell lysate samples (C). Relative protein expression in CM was

quantified (graph). (D and E) Images corresponding to Fig. 6L. Single-channel and merged

images of GFP-tagged STC2 (red), trans-Golgi marker TGN46 (green), and DAPI (blue) in H23

cells transfected with siRNAs against PI4KIIIβ (D) or GOLPH3 (E). Images taken before (T=0)

and 15 and 30 min after switching to a temperature (37˚C) that permits vesicles to exit the Golgi.

Scale bars: 3 μm.

A

E

D 0 min 15 min 30 min

siP

I4K

IIIβ

siC

TL

STC2-EGFP TGN46 STC2

TGN

DAPI

H23

STC2-EGFP TGN46 STC2

TGN

DAPI

STC2-EGFP TGN46 STC2

TGN

DAPI

STC2-EGFP TGN46 STC2

TGN

DAPI

STC2-EGFP TGN46 STC2

TGN

DAPI

STC2-EGFP TGN46 STC2

TGN

DAPI

H23

siG

OLP

H3

siC

TL

0 min 15 min 30 min

B C

CLU

HA (GOLPH3)

α-Tubulin

TIMP1

CLU

Vec GOLPH3

TIMP1

Rela

tive p

rote

in e

xpre

ssio

n

PRDX5

ANXA2

TIMP1

STC

2

SEM

A3C

CLU

PLO

D3

0

2

4

6 shCTLshPI4KIII#1shPI4KIII#2

**

**

****** ** **** **

*****

**P<0.01

***P<0.001

** **

Rela

tive p

rote

in e

xpre

ssio

n

TIMP1 CLU0

2

4

6

8

P<0.001

P=0.05 VecGOLPH3

Fig. S10. 1q amplification is associated with increased secretion. (A, B) Western blot

analysis of conditioned medium samples (CM) and cell lysates (A) and densitometric

quantification of the specific bands in (A) (B). (C) Protein concentrations in CM samples

determined by ELISA and normalized to the cell lysate values. Values expressed relative to

H3122 cells, which were set at 1. (D) Protein concentrations in CM samples determined by

ELISA.

H 2 1 2 2 H 1 2 9 9

0

5

1 0

1 5

Se

cre

ted

CL

U (

ng

/mL

)

D M S O

IN -9

P < 0 .0 0 1

P = 0 .0 5

H 2 1 2 2 H 1 2 9 9

0

2

4

6

8

Se

cre

ted

TIM

P1

(n

g/m

L)

D M S O

IN -9

P < 0 .0 0 1

P = 0 .0 2

A B C

CLU

STC2

TIMP1

PLOD3

H3122

H2122

H23

H1299

A549

H460

CM

Lysate

CLU

STC2

TIMP1

PLOD3

α-Tubulin

D

C L U S T C 2 T IM P 1 P L O D 3

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Re

lati

ve

se

cre

tio

n H 3 1 2 2

H 2 1 2 2

H 2 3

H 1 2 9 9

A 5 4 9

H 4 6 0

P = 0 .0 0 9 P = 0 .1 2

P = 0 .0 2 7 P = 0 .0 1 6

C L U T IM P 1

0 .0

0 .5

1 .0

1 .5

2 .0

Re

lati

ve

se

cre

tio

n

H 3 1 2 2

H 2 1 2 2

H 2 3

H 1 2 9 9

A 5 4 9

H 4 6 0

P = 0 .0 3

P = 0 .0 0 4

Fig. S11. PLOD3 maintains H2122 cell survival by activating MMP9. (A) qPCR analysis of

mRNA expression in H1299 cells transfected with the indicated siRNAs. (B) Relative cell

densities determined by WST-1 assays. (C) Anchorage-dependent colony formation by siRNA-

transfected H1299 cells. (D) Invaded cells in Transwell chambers. (E) Western blot detection of

apoptotic cells on the basis of PARP1 and caspase 3 cleavage. (F) Flow cytometric detection of

apoptotic cells. (G) Apoptotic cells detected by flow cytometry. Values expressed relative to

siCTL-transfected H1299_vector cells and H1299_PI4KIIIβ cells, which were set at 1. (H)

Detection of apoptotic H3122 cells by PARP1 western blot analysis (gels) and flow cytometry

(graph). (I) Western blot detection of apoptotic cells on the basis of PARP1 cleavage (arrow).

WT, wild type PLOD3; ∆GLT, glycosyltransferase-deficient PLOD3; ∆LH, lysyl hydroxylase-

A B C

D

α-Tubulin

PARP1

Cleaved PARP1

Cleaved caspase 3

H 1 2 9 9

% A

po

pto

tic

ce

lls

0

1

2

3

4

5

s iS T C 2

s iC T L

s iC L U

s iP L O D 3

s iT IM P 1

s iS E M A 3 C

**

* *P = 0 .0 0 4

H 1 2 9 9

Ce

ll p

roli

fera

tio

n (

OD

45

0)

D a y 1 D a y 2 D a y 3 D a y 4

0 .0

0 .3

0 .6

0 .9

1 .2 s iC T L

s iC L U

s iP L O D 3

s iT IM P 1

s iS E M A 3 C

s iS T C 2

****

** P < 0 .0 1

H 1 2 9 9

Re

lati

ve

co

lon

y n

um

be

r

0 .0

0 .5

1 .0

1 .5

***P < 0 .0 0 1

s iS T C 2

s iC T L

s iC L U

s iP L O D 3

s iT IM P 1

s iS E M A 3 C

*

***

*P < 0 .0 2

Re

lati

ve

ce

ll n

um

be

r

pe

r fi

eld

0 .0

0 .5

1 .0

1 .5 s iC T L

s iC L U

s iP L O D 3

s iT IM P 1

H 1 2 9 9

s iS E M A 3 C

s iS T C 2****

**

**P = 0 .0 0 4

***P < 0 .0 0 1

***

E F

G H

I J K

α-Tubulin

PARP1

Cleaved PARP1

H3122%

Ap

op

toti

c c

ell

s

siC

TL

siP

LO

D3

siT

IMP

1

0

5

1 0

1 5P = 0 .0 0 7

H 3 1 2 2

P = 0 .0 0 4

α-Tubulin

siCTL siPLOD3

PARP1

PLOD3

--

H2122

PLOD3:

H 2 1 2 2

% A

po

pto

tic

ce

lls

0

5

1 0

1 5 s iC T L

s iM M P 9 # 1

s iM M P 9 # 2

P < 0 .0 0 1

P < 0 .0 0 1

H 2 1 2 2

% A

po

pto

tic

ce

lls

0

3

6

9

1 2

P < 0 .0 0 1

P < 0 .0 0 1

P < 0 .0 0 1P L O D 3

C T L

s iC T L

s iP L O D 3

s iM M P 9

+ - -

- + +

- + -

siC

TL

siP

LO

D3#1

siP

LO

D3#2

siT

IMP

1#1

siT

IMP

1#2

0

5

1 0

1 5

2 0

H 2 1 2 2

Ac

tiv

e M

MP

9 (

ng

/mL

)

P = 0 .0 2

P = 0 .0 1

L

Re

lati

ve

ap

op

tos

is r

ate

siC

TL

siP

LO

D3

siT

IMP

1

0

2

4

6

V e c

P I4 K III

P = 0 .0 0 2

P = 0 .0 3

P = 0 .0 0 4

P = 0 .0 3

H 1 2 9 9

CLU PLOD3 TIMP1 SEMA3C STC20

1

2H1299

Rela

tive m

RN

A e

xpre

ssio

n

siCTLsiCLUsiPLOD3siTIMP1siSEMA3CsiSTC2

deficient PLOD3. (J) Flow cytometric detection of apoptotic cells. (K) Flow cytometric detection

of apoptotic H2122 cells after transfection with indicated siRNAs and treatment with

recombinant PLOD3 protein (PLOD3) or medium alone (CTL). (L) Quantification of active MMP9

in conditioned medium samples from H2122 cells transfected with the indicated siRNAs.

Fig. S12. PI4KIIIβ-dependent secretion regulates processes in the tumor

microenvironment. (A) Concentrations of secreted factors in conditioned medium samples. (B-

E) Total T cells (B), CD4+ T cell subsets (C), CD8+ T cell subsets (D), and myeloid cell subsets

(E) in 344SQ flank tumors. (F) Quantification of myeloid-derived suppressor cells

(CD45+CD11b+GR1+) in splenocytes co-cultured for the indicated amounts of time with PI4KIIIβ-

deficient or –replete 344SQ cells. Results expressed as percentages of total CD45+ cells.

s hC TL s hP I4 K B

0

2

4

6

8

T C e lls

% C

D3

+C

D4

5+

Ce

lls

P = 0 .6 2 5 0

s hC TL s hP I4 K B

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

C D 4

% C

D4

+ C

ell

s

P = 0 .0 9 7 1

s hC TL s hP I4 K B

3 0

4 0

5 0

6 0

7 0

8 0

C D 4 T R e g

% F

ox

P3

+C

D2

5+

Ce

lls

P = 0 .0 5 3 5

s hC TL s hP I4 K B

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

C D 4 IC O S

% i

CO

S+

Ce

lls

P = 0 .0 7 5 2

s hC TL s hP I4 K B

3 0

4 0

5 0

6 0

7 0

C D 8

% C

D8

+ C

ell

s

P = 0 .4 2 1 6

s hC TL s hP I4 K B

0

1 0

2 0

3 0

4 0

C D 8 E x h a u s te d

% P

D1

+ T

IM3

+C

ell

s

P = 0 .1 5 9 4

s hC T L s hP I4 K B

0

2 0

4 0

6 0

C D 8 M e m o ry + E ffe c to r

% C

D4

4h

i+ C

D6

2L

lo

w+

Ce

lls

P = 0 .0 9 4 0

s hC TL s hP I4 K B

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

C D 8 N a iv e

% C

D4

4lo

w+

CD

62

Lh

i+C

ell

s

P = 0 .6 3 2 9

s hC TL s hP I4 K B

0

2 0

4 0

6 0

8 0

M D S C

% C

D1

1b

+G

R1

+ P = 0 .0 1 4 4

s hC TL s hP I4 K B

0

1

2

3

4

D e n d ritic C e lls

% C

D1

1c

+ M

HC

II+

F4

/80

-G

R1

-

P = 0 .3 6 2 8

s hC TL s hP I4 K B

0

2 0

4 0

6 0

G ra n u lo c y te s

% F

4/8

0-C

D1

1b

+C

D1

1c

-

P = 0 .7 6 3 5

s hC TL s hP I4 K B

0

5

1 0

1 5

2 0

2 5

T o ta l M a c ro p h a g e s

% F

4/8

0+

CD

11

b+

GR

1-

P = 0 .2 5 6 3

A

B C

D

G-C

SF

Eo

tax

in

GM

-CS

F

IFN

g

IL-1

a

IL-1

b

IL-2

IL-4

IL-3

IL-5

IL-6

IL-7

IL-9

IL-1

0

IL-1

2 (

p4

0)

IL-1

2 (

p7

0)

LIF

IL-1

3

Lix

IL-1

5

IL-1

7

IP-1

0

KC

MC

P-1

MIP

-1a

MIP

-1b

M-C

SF

MIP

-2

MIG

RA

NT

ES

VE

G-F

TN

Fa

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4C

on

ce

ntr

ati

on

(p

g/m

l)s h C T L

s h P I4 K III

* * *

*

*P < 0 .0 5

**P < 0 .0 1

***P < 0 .0 0 1* * *

* *

*

344SQ

E

0 .5

1 .0

1 .5

S p le n o c y te c o -c u ltu re s

% C

D4

5+

CD

11

b+

GR

1+

4 8 h 9 6 h

s h C T L

s h P I4 K III

3 4 4 S Q

F

Supplementary Tables

Table S1. Clinical pathological characteristics of lung adenocarcinomas.

Variable Category Number Percentage

Adenocarcinomas 89

Age Average 66.3

Median (min –max) 66.0 (42 – 86)

Sex

Female 41 46%

Male 48 54%

Smoking Status

Current 39 44%

Former 39 44%

Never 11 12%

Race Caucasian 82 92%

Others 7 8%

Pathological T

T1 20 23%

T2 43 48%

T3 18 20%

T4 8 9%

Pathological N

N0 104 67%

N1 24 20%

N2 18 12%

Pathological Stage

I 37 42%

II 28 31%

III 24 27%

Neoadjuvant Tx Yes 0 0%

No 89 100%

Adjuvant

Yes 38 43%

No 49 55%

Unknown 2 2%

Vital status Alive 41 46%

Dead 48 54%

Recurrence No 54 61%

Yes 35 39%

Follow up Survival Months

Average 57.4

Median (Min Max) 55 (4 -144)

Table S2. Liquid chromatography–mass spectrometry of conditioned medium samples.

Accession Number Molecular Weight Fold change (shPI4KB/shCTL)

H2122 H23

SEMA3C_HUMAN 85 kDa 0 0.585588853

QPCT_HUMAN 41 kDa 0 0

PRP8_HUMAN 274 kDa 0 0.640054406

CATL2_HUMAN 37 kDa 0 0.688357307

RLA2_HUMAN 12 kDa 0 0.774060352

CLU_HUMAN 52 kDa 0 0.660924786

MYADM_HUMAN 35 kDa 0.091335958 0

MYH10_HUMAN 229 kDa 0.159884393 0.193414403

PPT1_HUMAN 34 kDa 0.184687752 0

EWS_HUMAN 68 kDa 0.198366827 0.575661876

SPTN2_HUMAN 271 kDa 0.209436227 0.484465541

MOT1_HUMAN 54 kDa 0.27817433 0.688357307

SPTB2_HUMAN 275 kDa 0.280028429 0.677483789

ARPC4_HUMAN 20 kDa 0.281483051 0.735328897

GP126_HUMAN 137 kDa 0.291541578 0.426033263

DAG1_HUMAN 97 kDa 0.29426331 0.553431212

ACTB_HUMAN 42 kDa 0.30786106 0.69159995

CYC_HUMAN 12 kDa 0.348650963 0.708871558

HEXB_HUMAN 63 kDa 0.38315582 0

CYTC_HUMAN 16 kDa 0.440819741 0.486102307

RL12_HUMAN 18 kDa 0.453056456 0.402137301

PLOD3_HUMAN 85 kDa 0.467250923 0.605559058

PRDX5_HUMAN 22 kDa 0.517292868 0.387393974

TPP1_HUMAN 61 kDa 0.519133719 0.688357307

PLOD1_HUMAN 84 kDa 0.539696943 0.536997535

MYH9_HUMAN 227 kDa 0.542067308 0.687811007

CAZA2_HUMAN 33 kDa 0.563627208 0.257748498

IBP2_HUMAN 35 kDa 0.563778256 0.419444248

ANXA2_HUMAN 39 kDa 0.616238562 0.609619783

BUB3_HUMAN 37 kDa 0.637616273 0.654562112

TIMP1_HUMAN 23 kDa 0.637656425 0.74650124

FBLN1_HUMAN 77 kDa 0.649654674 0.603874432

STC2_HUMAN 33 kDa 0.65898054 0.487150645

Table S3. Primers.

qPCR primers

Gene Forward (5′-3′) Reverse (5′-3′)

mRNA

PI4KB (human) TGGTCGGTGGATGACATAGGCG CTGGTGATGCTGTCCACAGAGA

VPS45 (human) CAGCAATAGCCTGCCAGGACTA GTGATAGCCACAGCATCTTTGGG

GOLPH3L (human) CTCTGAAACACATCAAAGCAACTG CTTTGCGATGCGCTCTCGTACA

RAB13 (human) GACATCTTGCTCAAGTCAGGAGG CAGGGAGCACTTGTTGGTGTTC

FAPP1 (human) CTTCGCCTCTACTGTGACCTCT CGTGGCACTAAGCAGAGAAGAG

CERT (human) TTGGTAAGCCCACCAGAGGGAA GGATACTCTCGCTTTGCCACTG

OSBP (human) CTGGACCGATTAGAGGAGAATGG TTTCCTGACGCAATGTCCAGCC

BLZF1 (human) TACAGTGTGATGTATGGCGAAGTA CGTGTGCATCACGGTTTTGACG

KLHL20 (human) CAAGTTCCTGGTCGGCACAGTA AGGTTTCCGTGGTCTCGTCCTT

VAMP4 (human) GCTTATCGGATAATGCAACAGCTT GCAGCAACCAAAGCCATGATGG

KLHL12 (human) CCAAGACTCAGGAGTGGAGCTT GACATTCCACTGAACTAAGGCGG

GOLTA1 (human) ACGGCACAAACTCAAGGGAACC AGACATTGCCCAGGAAGCCGAA

STX6 (human) CACGAATTGGAGAGCACTCAGTC GAGGATGAGCACAACCAACAGG

RFWD2 (human) GTCAGTGAGGATAGCACAGTGC GAGAACTGCCACTGAAACCTGG

QSOX1 (human) ACATGGCTGACCTGGAATCTGC GCAGGAAGTTCTGGACTAAGGG

RAB29 (human) TGCTCTGAAGGTTCTCCAGTGG GGCAGAGGCATCCCGATAATAC

ACBD3 (human) TCAGTGAGTCCAGCGATGACGA GGCACAATCTCATCCAGCAAAGG

NMNAT2 (human) GTAGTGACCTGCTGGAGTCCTT ATGATTCGGTCTGTGTCGGCTG

ARF1 (human) CCTCCTGGTGTTCGCCAACAAG TCCAGTCCTTCATAGAGCCCGT

SCAMP3 (human) GGATGTGCTCTTTGTCCTCCAG GCACAGCAATGCCAGTGAAGAG

GPR89A (human) ATCCCTTTCCCATTCTCAGCCC GGCAGTTGACAGCACCAAATCC

TMEM79 (human) CAGGATACCCTCAAACTGCTCC CATCGACAGCAGTGGCAGAAAC

VANGL2 (human) AGCCTCAGTTCACGCTCAAGGT GGGTTGTAGACAGGGAAGTCATG

SEMA3C (human) ACCCACTGACTCAATGCAGAGG CAGCCACTTGATAGATGCCTGC

STC2 (human) GCATGACTTTTCTGCACAACGCT GGCTTATGCAGCCGAACCTGTG

TIMP1 (human) GGAGAGTGTCTGCGGATACTTC GCAGGTAGTGATGTGCAAGAGTC

CLU (human) TGCGGATGAAGGACCAGTGTGA TTTCCTGGTCAACCTCTCAGCG

PLOD3 (human) CGAGTGTGAGTTCTACTTCAGCC CCAGAAGTTGGACCACAGCTTG

MMP9 (human) GCCACTACTGTGCCTTTGAGTC CCCTCAGAGAATCGCCAGTACT

Genomic DNA

gPI4KB (human) GGAATCGGTAGGGTCCTGAC CCTTCAGGCTGCCACAAG

gGOLPH3L (human) CAGGTGAGAATGACCACTTTAACTC CTCTTTATCTTTTAGTCCCAGAAGCA

gRAB13 (human) GGCCCACTAGTTAATTCACATAGACT AGGGAAAAGGTGACGAAAATTC

gVPS45 (human) GCGTTTGTTGAGAATTATCCACA CTGGAGAGCACTAGAATGGTCAT

gRPL32 (human) CGTAACTGGCGGAAACCC GTTGCACATCAGCAGCACTT

Constructs (pEGFP-C3)

PI4KB (mouse) CCGCTCGAGATGGACTACAAAGACGATGACGACAAGGGATCAGGTTCCGGGTCTATGGGAGACATGGTAGTGGAG

CGGGATCCTCACATGATGCCATTGGTG

STC2 (human) GGAATTCTGTGTGCCGAGCGGCT CGGGATCCTCACCTCCGGATATCAGAATAC

Constructs (pLVX)

PLOD3 (mouse) GCACGCACTCTAGAGCCACCATGGCTGCGGCAGGCCCGGAACCC GCACGCACGCGGCCGCTTAGGGGTCAACAAAGGACACCATG

LH MT CATGTTTGTGGACAGCTACGCCGTGATTCTGGCAAGCAG CTGCTTGCCAGAATCACGGCGTAGCTGTCCACAAACATG

GLTMT GCTTCGGCCACACCATGCCTCGTCCACCTTCACTCTC GAGAGTGAAGGTGGACGAGGCATGGTGTGGCCGAAGC

Genotyping primers

Kras LSL-G12D (mouse)

GTCTTTCCCCAGCACAGTGC CTCTTGCCTACGCCACCAGCTC