Developmental Cell, Volume 37

Supplemental Information

A Drosophila Genome-Wide Screen Identifies

Regulators of Steroid Hormone Production

and Developmental Timing

E. Thomas Danielsen, Morten E. Moeller, Naoki Yamanaka, Qiuxiang Ou, Janne M.Laursen, Caecilie Soenderholm, Ran Zhuo, Brian Phelps, Kevin Tang, Jie Zeng, ShuKondo, Christian H. Nielsen, Eva B. Harvald, Nils J. Faergeman, Macy J. Haley, Kyle A.O'Connor, Kirst King-Jones, Michael B. O'Connor, and Kim F. Rewitz

Supplemental Figures

Figure S1 (related to Figure 1). (A) Gene ontology (GO) analysis of the gene set showing the top enriched

functional categories. Genes were grouped into common functional categories based on GO terms from both

Drosophila genes and their human orthologs. (B) Venn diagrams showing overlap between enriched GO

terms of gene sets associated with different phenotypes. Numbers indicate total number of GO terms for each

gene set.

Figure S2 (related to Figure 2). (A) Phylogenetic tree showing relationship between the Drosophila Sit

protein and human very long chain fatty acid elongase (ELOVL) proteins. Neighbor-joining tree is based on

a ClustalW multiple alignment. Hsap; Homo sapiens. Dmel; Drosophila melanogaster. (B) sit mRNA levels

in various tissues from larvae (whole body, fat body, midgut and brain) and adults (ovary, testis, accessory

gland). (C) Effect of PG-specific sit knockdown on pupal size. (D) Schematic representation of the sit

genomic locus with the 1325 bp CRISPR/Cas9 induced deletion that removes the sit coding region

generating the sit1D

and sit2C

alleles. (E) Nucleotides deleted in the sit1D

and sit2C

mutants compared to wild

type. The 20 bp sgRNA sequences (green) and the protospacer adjacent motif (PAM: blue) are indicated. (F)

Survival of sit1D

and sit2C

mutants. (G) Quantification shows that animals with RNAi knockdown of sit and

Npc1a in PG and the sit loss-of-function mutant have significantly increased number of the lipid droplets in

PG cells. CARS microscopy images of PG (H) fat body and crop (I) of sit mutants. (J) Cholesterol ester

levels in L3 larvae with ubiquitous RNAi mediated silencing of Npc1a or sit. RNAi knockdown was

conditionally induced in L2 larvae 96 hours after egg lay (AEL) by shifting larvae from 18oC to 29

oC and

assayed two days later. wt; wild type. Detailed description of genotypes is given in Supplemental

Experimental Procedures. Error bars indicate SEM (***P<0.001).

Figure S3 (related to Figure 2). (A) A high cholesterol diet (+) rescues the L1 arrest phenotype otherwise

caused by Npc1a-RNAi in the PG. Five day old larvae. (B) Addition of 40 µg/ml cholesterol to the diet (+

cholesterol) rescues the pupariation delay caused by knock down of sit in the PG. (C) Ex vivo incubation of

ring glands with NBD-cholesterol. RNAi induced silencing of either sit or the ceramide synthase schlank in

the PG results in accumulation of NBD-cholesterol. (D) Quantification of lipid droplets in PG cells with

reduced expression of schlank compared to the control. (E) Analysis of ceramide (Cer) levels in L3 larvae

using LC-MS. RNAi was induced conditionally in L2 and ceramide levels were determined in L3 larvae 48

hours later. (F) Knockdown of sit or schlank in PG blocks trafficking and degradation of GFP-LAMP

causing an accumulation. Dotted white lines encircle the PG cells. (G) Rab7 (late endosome marker) vesicles

of PG cells from animals with reduced expression sit and schlank. (H) Quantification shows an increased

frequency of enlarged Rab7 vesicles in PG cells with reduced expression of sit or schlank. Detailed

description of genotypes is given in Supplemental Experimental Procedures. AEL; after egg lay. Error bars

indicate SEM (*P<0.05; **P<0.01; ***P<0.001).

Figure S4 (related to Figure 4 and Figure 6). (A) Quantification of lipid droplets in PG cells from CARS

images. Inhibition of TOR signaling by overexpression of TSC1/2 in the PG reduces number of lipid

droplets, while inhibition (Akt-RNAi) or stimulation (InR) of insulin signaling has no effect. (B) Blocking

ecdysone signaling in the PG cells by overexpression of EcRDN

or by knockdown of EcR (EcR-RNAi) results

in an increase of lipid droplets in PG cells. (C) Quantification of lipid droplets from CARS images.

Inhibition of TOR signaling by overexpression of TSC1/2 prevents accumulation of lipid droplets in Npc1a-

RNAi PG cells. Detailed description of genotypes is given in Supplemental Experimental Procedures. Error

bars indicate SEM (***P<0.001).

Supplemental Tables

Table S1. Primary screen results (related to Figure 1). All genes screened for effects of PG-specific

knockdown on developmental progression. Transgenic RNAi lines (see transformant ID) targeting 12,504

Drosophila genes were used to specifically knockdown genes in the PG using the phm-Gal4 driver.

Knockdown of 1,906 genes in the PG caused developmental arrest and delay phenotypes. NOP; no obvious

phenotype.

Table S2. Gene Ontology (GO) analysis (related to Figure 1). GO term enrichment analysis of gene hits

identified the primary screen. GO terms from both Drosophila genes and their human orthologs were

included.

Supplemental Experimental Procedures

Developmental timing assays, temperature shift experiments, starvation and cholesterol

treatment

For developmental timing analysis, larvae were synchronized by allowing flies to deposit eggs at 25˚C for 4

hours in a humid chamber on apple juice agar plates supplemented with yeast paste. Newly hatched L1

larvae were collected 24 hours later, transferred to standard food (25 larvae/vial). For rescue experiments

with dietary cholesterol, the food was supplemented with 40 μg/ml cholesterol dissolved in ethanol.

Conditional induction of RNAi or overexpression was done using the temperature dependent tub-Gal80ts.

Eggs were collected at 25˚C on apple juice agar plates supplemented with yeast for four hours and incubated

at 18˚C (permissive temperature). L1 larvae were collected 48 hours later and transferred into vials (25

larvae/vial) and reared at 18°C for two days before shifted to 29°C (restrictive temperature) for two

additional days. For starvation, synchronized larvae were raised on normal standard food at 25˚C and

transferred 92 hours AEL to either normal food or 0.8% agar (starvation condition). Larvae were reared

under these different conditions for 10 hours before collected for analyses.

GO term analysis

All genes were annotated with GO terms, and to increase the coverage of GO annotations on the genes in the

dataset, GO terms for the human orthologs of the Drosophila genes were added to the gene annotation (using

Ensembl 79 HomoSapiensGRCh38.p2). Obsolete GO terms were excluded from the analysis. GO terms were

assigned to functional categories based on results from QuickGo (https://www.ebi.ac.uk/QuickGO/) and then

manually curated. Regardless of the number and categorization of the GO terms of a gene, a single gene only

contributed at most once to any functional category. Significantly enriched GO terms in genes associated

with developmental defects were assessed with Fischer's exact test with Bonferroni correction for multiple

testing. Analyses were performed using Unix command line tools, Python v 2.7.2 and R v. 3.0.3.

Ecdysteroid measurements

Ecdysteroid levels were measured with a commercial ELISA kit (ACE Enzyme Immunoassay, Cayman

Chemicals) which detects ecdysone and 20-hydroxyecdysone (20E) with the similar affinity (Porcheron et

al., 1976). Briefly, ecdysteroids were extracted from whole animals at the designated time points by

homogenization in 0.5 ml methanol by a close fitting pestle, followed by centrifugation at 14,000 g and

collection of the supernatant. The remaining tissues were re-extracted using the same procedure; first with

0.5 ml methanol and then with 0.5 ml ethanol. The supernatants were pooled and 0.5 ml was evaporated

using a SpeedVac followed by re-suspension in ELISA buffer (1 M phosphate solution, 1% BSA, 4 M

sodium chloride and 10 mM EDTA). The ELISA was performed according to the manufacturer’s protocol,

with a standard curve of 20E (Sigma). Absorbance was measured at 405 nm on a plate reader, ELx80

(BioTek) using Gen5 software (BioTek).

Pupal size measurements

For analysis of pupal size, pupae were aligned on a cover slide and imaged using Zeiss AxioCam ICc 1

camera mounted on a Zeiss AxioZoom.V16 microscope. Images of a total of 30-40 pupae per genotype were

processed using homemade Matlab software that determines pupal size kindly provided by Pierre Leopold.

Phylogenetic tree

Phylogenetic relationships of Drosophila Sit and the human ELOVL family of proteins were performed by

generating a neighbor-joining tree based on multiple alignments of the entire amino acid sequences of

Drosophila Sit and human ELOVL proteins using ClustalW (Larkin et al., 2007).

Ex vivo NBD-cholesterol incubation assay

Analysis of fluorescent NBD-cholesterol uptake in tissues ex vivo was performed as described (Enya et al.,

2014). Brain-ring gland complexes (BRGC) from feeding third instar larvae were dissected in Schneider’s

Drosophila Medium (Gibco) containing 10% Fetal Bovine Serum (Gibco), 100 U/ml penicillin and 100

µg/ml streptomycin (Gibco). Exposure to light was avoided due to light-sensitivity of NBD-cholesterol

(Molecular Probes). Tissues were transferred to fresh medium containing 0.5% 10 µM NDB-cholesterol

(dissolved in 96% ethanol) and incubated at room temperature for six hours in dark and humid conditions

under gentle shaking. For co-localization of Atg8a vesicles and NBD-cholesterol, tissues were incubated

under these conditions for 8 hours. After the incubation, samples were washed twice with PBS followed by

fixation in 4% formaldehyde for 20 min at room temperature. The fixed tissues were washed three times with

PBS, mounted in PBS and immediately imaged using a Zeiss LSM 710 confocal microscope. Images were

processed and analyzed using ImageJ (NIH).

Analysis of cholesterol levels

Total lipids were extracted from whole larvae in a solution of 100 µl H2O, 125 µl chloroform and 250 µl

methanol and sonicated for 10 minutes at medium intensity using a Biorupter (Diagenode). Chloroform (125

µl) and 0.2 M KCL (125 µl) was added and samples were vortexed and centrifuged for 10 min at 2,000 rpm

at room temperature. The bottom phase was transferred to a new tube for further lipid analysis. The top

phase was re-extracted with 100 µl chloroform, vortexed, centrifuged and the bottom phase was transferred

and combined with the first bottom phase. The interphase was lyophilized and re-suspended in H2O and used

to determine protein levels using standard Bradford protein assay. The combined bottom phases were dried

under a stream of N2, re-suspended in chloroform/methanol (1:1) and examined by high performance thin

layer chromatography (HP-TLC) using cholesterol as standard. Band intensities were determined by

densiometry and normalized to amount of protein.

Extraction and quantification of ceramides in Drosophila larvae

Lipids were extracted from 5 larvae as previously described (Fong et al., 2009) except from the lower phase

was used for ceramide analyses. Prior to extraction ceramide-d18:1/17:0 was added as internal standard. The

lower phase was dried down under flow of nitrogen gas and resuspended in isopropanol/methanol/H2O

(5:1:4) with 5 mM NH4OAc and 0.1 % CH3COOH (mobile phase A). Ceramide species were quantified by

LC-MS. Extracted samples (5 μl) were injected on to an Agilent 1290-LC and separated on an Agilent

Zorbax Extended C18 rapid resolution column (2.1x150 mm, 1.8 μm) kept at 50 °C. Lipids were resolved

using a gradient starting from 100% mobile phase A at a flow rate of 0.350 ml/min for 3 min, to 20% mobile

phase B (isopropanol/H2O (99/1) with 5 mM NH4OAc and 0.1 % CH3COOH over 2 min at a linear gradient,

then to 30% over 20 min, then to 95% mobile phase B over 10 min, and then with 95% mobile phase B for 1

min. Ceramides were detected on an Agilent 6530 Accurate-Mass QTOF mass spectrometer equipped with a

Jetstream ESI ion source, which was operated in positive mode. Instrument parameters were set as follows:

sheath gas temperature, 350°C; sheath gas flow, 12 L/min; nebulizer, 35 psi; dry gas temperature, 300°C; dry

gas flow, 11 L/min; and capillary entrance voltage, 3500 V. Fragmentor and skimmer was operated at 125 V

and 65 V, respectively. The MS scan data were collected at a rate of 4 spectra/s in the range of m/z 100–

1500. The m/z of all ions in the mass spectra were corrected by two reference ions m/z 121.050873 and m/z

922.009798 (Agilent). Data were collected with MassHunter Data Acquisition ver. B.06.01 (Agilent), and

MassHunter Qualitative Analysis ver. B.07.00 (Agilent) was applied to identify lipid species. Peak areas of

the internal standard and ceramides were integrated from extracted ion chromatograms (EIC) by Profinder

B.06.00 (Agilent).

Immunostaining

Tissues were dissected in PBS followed by fixation in 4% formaldehyde for 25 minutes at room temperature.

After fixation, samples were washed with PBS containing 0.1% Triton X100 (PBT). Tissues were blocked

for 1 hour in PBT containing 5% normal goat serum (NGS) and incubated overnight at 4˚C with primary

antibodies diluted in PBT with 5% NGS. Tissues were then washed in PBT and incubated at room

temperature with secondary antibodies diluted in PBT with 5% NGS for 2 hours. The following primary

antibodies were used: guinea pig anti-LpR2 1:100 a generous gift from Joaquim Culi (Parra-Peralbo and

Culi, 2011), rabbit anti-Phm 1:200 (Parvy et al., 2005), rabbit anti-GFP 1:200 (MBL, #598), mouse anti-GFP

1:200 (Clontech, #632380). The following fluorescent conjugated secondary antibodies were used in this

study: goat anti-rabbit Alexa Fluor 555 (Invitrogen, #A21429), goat anti-mouse Alexa Fluor 488 (Invitrogen,

#A11001), goat anti-guinea pig Alexa Fluor 555 (Invitrogen, #A21435). Tissues were then washed and

mounted in ProLong Gold Antifade (Molecular Probes). Imaging was performed using a Zeiss LSM 710

confocal microscope. Images were further processed and analyzed using ImageJ (NIH) software.

Western blotting

For Western blot analysis, five whole larvae were rinsed in PBS and homogenized in 100 µl Laemeli Sample

Buffer (Bio-Rad) containing 2-mercaptoethanol. Tissue samples were boiled for five minutes and cleared for

debris by centrifugation at 14,000 g for three minutes. Proteins from the samples were resolved on a 4-20%

polyacrylamide gradient gel (Bio-Rad) and transferred onto a PVDF membrane (Millipore). Primary

antibodies used were: guinea pig anti-LpR2 1:1000, rabbit anti-GFP 1:1000, and mouse anti-tubulin 1:5000

(Sigma Aldrich, #T9026). Primary antibodies were detected using the IRDye 680RD and 800CW infrared

dye secondary antibodies 1:10,000 (LI-COR) and membranes were imaged on an Odessey Fc (LI-COR) and

quantified using the Image Studio software.

Quantitative RT-PCR

For gene expression analysis using quantitative RT-PCR (qPCR) five whole larvae or pooled tissues were

used for each replicate. Extraction of total RNA was performed using RNeasy mini kit (Qiagen) according to

the manufacturer’s instructions. The RNA was treated with DNase (Qiagen) and quantified using NanoDrop

(Thermo Scientific) before converted into cDNA using iScript Reverse Transcription Supermix (Bio-Rad).

Relative gene expression was measured with Quantitect SYBR Green PCR Kit (Qiagen) using Mx3005P

qPCR System (Agilent Technologies). Melting curves for each primer pair were validated to ensure

amplification of a single PCR product. Primers used for qPCR were designed using the Primer3

(Untergasser et al., 2012) and are listed below. Gene expression was normalized to the reference genes,

Rpl23 and Rpl32, as previous described (Danielsen et al., 2014).

List of oligonucleotides used for qPCR

Target gene Forward primer 5’-3’ Reverse primer 5’-3’

sit (CG5278) GTCAACGCAACACAGGTGGA ACGAAGAAGAGATAGAAGGCCAG

Npc1a TTGCAACCAAGCAGTTAGCA CAATTTTGAAGGGCTCTGGA

LpR1 TGGCGGTAAGGATGGTACAC GACCGACTGGGACAAACAG

LpR2 GAAATAGCCTTGCATGTGATTGC GTGGTAGACGGGATTCTCGAA

Rpl23 GACAACACCGGAGCCAAGAACC GTTTGCGCTGCCGAATAACCAC

Rpl32 TAAGCTGTCGCACAAATGGCG AACGCGGTTCTGCATGAGCA

Genotypes

Figure Name Genotype

Figure 1

B Control

UAS-RNAi lines

w; +; phm-Gal4/ +

w; UAS-RNAi lines/+; phm-Gal4/+

Figure 2

B w1118

C-E

Control

sit-RNAi

w; +; phm-Gal4/ +

w; UAS-sit-RNAi/+; phm-Gal4/ +

F

Control

sit-RNAi

sit1D/2C

Npc1a-RNAi

schlank-RNAi

w; +; phm-Gal4, UAS-GFP/ +

w; UAS-sit-RNAi/+; phm-Gal4, UAS-GFP/ +

+; +; Sit1D

/ Sit2C

w; tub-Gal80ts/ UAS-Npc1a-RNAi

KK; phm-Gal4, UAS-GFP/ +

w; UAS-Schlank-RNAi/+; phm-Gal4, UAS-GFP/ +

G w; UAS-sit-RNAi/+; phm-Gal4/ +

H

Control

Npc1a-RNAi

sit-RNAi

w; Act-Gal4/ +; tub-Gal80ts/ +

w; Act-Gal4/ UAS-Npc1aKK

-RNAi; tub-Gal80ts/ +

w; Act-Gal4/ UAS-sit-RNAi; tub-Gal80ts/ +

I

Control

Npc1a-RNA

sit-HA

w; +; phm-Gal4/ +

w; +; phm-Gal4/ UAS-Npc1aGD

-RNAi

w; UAS-sit-HA/+; phm-Gal4/ UAS-Npc1aGD

-RNAi

Figure 3

A w1118

B

Control

InR

Rheb

w; +; phm-Gal4, UAS-GFP/ +

w; +; phm-Gal4, UAS-GFP/ UAS-InR29.4

w; +; phm-Gal4, UAS-GFP/ UAS-Rheb

C

Control

Rheb

EcRDN

w; +; phm-Gal4, Sit-venus/ +

w; +; phm-Gal4, Sit-venus/ UAS-Rheb

w; UAS-EcRDN

/ +; phm-Gal4, sit-venus/ +

D

Control

EcRDN

w; +; phm-Gal4, UAS-GFP/ +

w; UAS-EcRDN

/ +; phm-Gal4, UAS-GFP/ +

E

Control

Rheb

EcRDN

w; +; phm-Gal4, UAS-GFP/ +

w; +; phm-Gal4, UAS-GFP/ UAS-Rheb

w; UAS-EcRDN

/ +; phm-Gal4, UAS-GFP/ +

Figure 4

A

Control

Akt-RNAi

InR

TSC1/2

EcRDN

EcR-RNAi

w; tub-Gal80ts/ +; phm-Gal4, UAS-GFP/ +

w; tub-Gal80ts/UAS-Akt1-RNAi; phm-Gal4, UAS-GFP/ +

w; tub-Gal80ts/ +; phm-Gal4, UAS-GFP/ UAS-InR

29.4

w; tub-Gal80ts/ +; phm-Gal4, UAS-GFP/ UAS-TSC-1, UAS-TSC-2

w; UAS-EcRDN

/ +; phm-Gal4, UAS-GFP/ +

w; UAS-EcR-RNAi/ +; phm-Gal4, UAS-GFP/ +

B-D

Control

EcRDN

w; +; phm-Gal4/ +

w; UAS-EcRDN

/ +; phm-Gal4/ +

E and F Control

Rheb

w; UAS-mCherry-Atg8a/ +; phm-Gal4/ +

w; UAS-mCherry-Atg8a/ +; phm-Gal4/ UAS-Rheb

Figure 5

A and

B

Control

Atg1-RNAi

Atg7-RNAi

Atg8a-RNAi

Atg8aKG07569

w; +; phm-Gal4, UAS-GFP/ +

w; UAS-Atg1-RNAi/ +; phm-Gal4, UAS-GFP/ +

w; UAS-Atg7-RNAi/ +; phm-Gal4, UAS-GFP/ +

w; UAS-Atg8a-RNAi/ +; phm-Gal4, UAS-GFP/ +

Atg8aKG07569

(X chromosome); +; +; +

C w; UAS-mCherry-Atg8a/ +; phm-Gal4/ +

D w1118

E w; +; Sit-venus

Figure 6

A

Control

Npc1a-RNAi

TSC1/2

Npc1a-RNAi;

TSC1/2

w; +; P0206-Gal4, UAS-mCD8::GFP/ +

w; +; P0206-Gal4, UAS-mCD8::GFP/ UAS-Npc1aGD

-RNAi

w; +; P0206-Gal4, UAS-mCD8::GFP/ UAS-TSC-1, UAS-TSC-2

w; +; P0206-Gal4, UAS-mCD8::GFP/ UAS-Npc1aGD

-RNAi, UAS-TSC-1,

UAS-TSC-2

B and C

Control

Npc1a-RNAi

Atg1/13; Npc1a-

RNAi

w; +; P0206-Gal4, UAS-mCD8::GFP/ +

w; +; P0206-Gal4, UAS-mCD8::GFP/ UAS-Npc1aGD

-RNAi

w; UAS-Atg1, UAS-Atg13/+; P0206-Gal4, UAS-mCD8::GFP/ UAS-

Npc1aGD

-RNAi

Figure S2

B w1118

C

Control

sit-RNAi

w; +; phm-Gal4/ +

w; UAS-sit-RNAi/+; phm-Gal4/ +

F

sit1D

sit2c

sit1D/2c

+; +; sit1D

/sit1D

+; +; sit2C

/sit2C

+; +; sit1D

/sit2C

G

Control (left panel)

sit-RNAi

sit-/sit

-

Control (right

panel)

Npc1a-RNAi

w; +; phm-Gal4, UAS-GFP/ +

w; UAS-sit-RNAi/+; phm-Gal4, UAS-GFP/ +

+; +; sit-/sit

-

w; tub-Gal80ts/ +; phm-Gal4, UAS-GFP/ +

w; tub-Gal80ts/ UAS-Npc1a-RNAi

KK; phm-Gal4, UAS-GFP/ +

H

Control

sit1D

sit2C

y2 cho

2 v

1; +; +

+; +; sit1D

/sit1D

+; +; sit2C

/sit2C

I

Control

sit1D/2c

y2 cho

2 v

1; +; +

+; +; sit1D

/sit2C

J

Control

Npc1a-RNAi

sit-RNAi

w; Act-Gal4/ +; tub-Gal80ts/ +

w; Act-Gal4/ UAS-Npc1aKK

-RNAi; tub-Gal80ts/ +

w; Act-Gal4/ UAS-sit-RNAi; tub-Gal80ts/ +

Figure S3

A

Control

Npc1a-RNAi

w; +; phm-Gal4/ +

w; UAS-Npc1aKK

-RNAi/+; phm-Gal4/ +

B

Control

sit-RNAi

w; +; phm-Gal4/ +

w; UAS-sit-RNAi/+; phm-Gal4/ +

C

Control

sit-RNAi

schlank-RNAi

w; +; phm-Gal4/ +

w; UAS-sit-RNAi/+; phm-Gal4/ +

w; UAS-schlank-RNAi/+; phm-Gal4/ +

D

Control

schlank-RNAi

w; +; phm-Gal4, UAS-GFP/ +

w; UAS-schlank-RNAi/+; phm-Gal4, UAS-GFP/ +

E

Control

sit-RNAi

w; Act-Gal4/ +; tub-Gal80ts/ +

w; Act-Gal4/ UAS-sit-RNAi; tub-Gal80ts/ +

F

Control

sit-RNAi

schlank-RNAi

w; αtub-GFP-LAMP/ +; phm-Gal4/ +

w; UAS-sit-RNAi/ αtub-GFP-LAMP; phm-Gal4/ +

w; UAS-schlank-RNAi/ αtub-GFP-LAMP; phm-Gal4/ +

G and

H

Control

sit-RNAi

schlank-RNAi

w; UAS-Rab7-GFP/ +; phm-Gal4/ +

w; UAS-sit-RNAi/ UAS-Rab7-GFP; phm-Gal4/ +

w; UAS-schlank-RNAi/ UAS-Rab7-GFP; phm-Gal4/ +

Figure S4

A

Control

Akt-RNAi

InR

TSC1/2

w; tub-Gal80ts/ +; phm-Gal4, UAS-GFP/ +

w; tub-Gal80ts/UAS-Akt1-RNAi; phm-Gal4, UAS-GFP/ +

w; tub-Gal80ts/ +; phm-Gal4, UAS-GFP/ UAS-InR

29.4

w; tub-Gal80ts/ +; phm-Gal4, UAS-GFP/ UAS-TSC-1, UAS-TSC-2

B

Control

EcRDN

EcR-RNAi

w; +; phm-Gal4, UAS-GFP/ +

w; UAS-EcRDN

/ +; phm-Gal4, UAS-GFP/ +

w; UAS-EcR-RNAi/ +; phm-Gal4, UAS-GFP/ +

C

Control

Npc1a-RNAi

TSC1/2

Npc1a-RNAi;

TSC1/2

w; +; P0206-Gal4, UAS-mCD8::GFP/ +

w; +; P0206-Gal4, UAS-mCD8::GFP/ UAS-Npc1aGD

-RNAi

w; +; P0206-Gal4, UAS-mCD8::GFP/ UAS-TSC-1, UAS-TSC-2

w; +; P0206-Gal4, UAS-mCD8::GFP/ UAS-Npc1aGD

-RNAi, UAS-TSC-1,

UAS-TSC-2

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