www.sciencetranslationalmedicine.org/cgi/content/full/6/221/221ra15/DC1

Supplementary Materials for

Antioxidants Accelerate Lung Cancer Progression in Mice

Volkan I. Sayin, Mohamed X. Ibrahim, Erik Larsson, Jonas A. Nilsson, Per Lindahl,* Martin O. Bergo*

*Corresponding author. E-mail: martin.bergo@gu.se (M.O.B.); per.lindahl@wlab.gu.se (P.L.)

Published 29 January 2014, Sci. Transl. Med. 6, 221ra15 (2014) DOI: 10.1126/scitranslmed.3007653

This PDF file includes:

Fig. S1. Antioxidant supplementation increases tumor stage in mice with B-RAFV600E–induced lung cancer. Fig. S2. Administration of antioxidants to mice with K-RASG12D–induced lung cancer reduces tumor expression of endogenous antioxidant genes. Fig. S3. NAC and vitamin E reduce ROS and DNA damage and increase tumor cell proliferation. Fig. S4. Antioxidants do not affect the amounts of apoptotic and senescent cells in tumors of mice with K-RASG12D– and B-RAFV600E–induced lung cancer. Fig. S5. NAC and Trolox increase the proliferation of fibroblasts expressing oncogenic, but not wild-type, K-RAS and B-RAF. Fig. S6. Antioxidants increase the proliferation of MYC-transformed fibroblasts. Fig. S7. Antioxidants increase the proliferation of human lung cancer cell lines expressing wild-type p53. Fig. S8. TP53 is required for antioxidants to increase the proliferation of human lung cancer cell lines. Reference (41)

Other Supplementary Material for this manuscript includes the following: (available at www.sciencetranslationalmedicine.org/cgi/content/full/6/221/221ra15/DC1)

Table S1. Original data (Excel spreadsheet). Table S2. Exact P values (Excel spreadsheet).

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Fig. S1. Antioxidant supplementation increases tumor stage in mice with B-RAFV600E–induced lung

cancer. (A) Tumor stage in lungs from mice with B-RAFV600E-induced lung cancer (n = 717–1725

tumors in lungs from 10–16 mice/group). P values are for comparisons of the percentage of stage 3

tumors in antioxidant-treated and control mice. (B) Representative tumors in hematoxylin and eosin–

stained lung sections. Scale bar, 500 µm. **** P < 10–10. Exact P values are provided in Table S2.

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Fig. S2. Administration of antioxidants to mice with K-RASG12D–induced lung cancer reduces

tumor expression of endogenous antioxidant genes. (A) Principal component (PC) analysis of

transcriptome sequencing profiles of tumors from NAC- and vitamin E–treated and control mice (n =

30 tumors; 10/group). From each sample, 27.1–35.4 × 106 read-pairs were generated, resulting in

1.89 × 109 total reads. (B) Upper and lower panels, unbiased pathway analyses based on BioCarta,

KEGG, Reactome, and GO gene sets revealed coordinated downregulation of genes involved in the

endogenous ROS defense system. No notable gene categories were identified among upregulated

genes. (C) TaqMan analyses of RNA from an independent set of tumors confirming reduced

expression of the 8 selected genes shown in Figure 1H (n = 5 tumors from 5 mice/group). (D)

TaqMan analyses showing reduced expression of endogenous antioxidant genes in normal lung

tissue of NAC- and vitamin E–treated wild-type mice (n = 4 mice/group). (E) NRF2 target genes

(red data points), defined previously by ChIP-seq and expression profiling (21), were enriched

among the 144 genes that were significantly repressed by both antioxidants (blue data points).

However, only a minority of repressed genes were predicted targets of NRF2 (9 of the 144 repressed

genes). (F) P53 target genes (red) (41) were not enriched in the gene set (blue). * P < 0.05. Exact P

values are provided in Table S2. Data on graphs are presented as mean ± SEM.

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Fig. S3. NAC and vitamin E reduce ROS and DNA damage and increase tumor cell proliferation. (A)

Left panel, quantification of ROS in lung sections of antioxidant-treated and control B-RAFV600E

mice as judged by DCF fluorescence (n = 5 fields-of-view/lung; 5 lungs/group for NLT; n = 25

tumors from 5 mice for Ctrl, NAC, and Vit E). Right panels, representative micrographs showing

DCF-fluorescence. (B) Antioxidants increase the ratio between reduced (GSH) and oxidized (GSSG)

forms of glutathione in tumors (n = 4 mice; 1 tumor/mouse). (C) Left panel, quantification of 8-

oxoguanine-positive cells in lungs of antioxidant-treated and control B-RAFV600E mice (n = 2–3

tumors/lung; 10 lungs/group). Right panels, representative immunohistochemistry micrographs

showing 8-oxoguanine staining. (D) Left panels, quantification of phosphorylated histone 3–positive

cells in lung tumors (n = 5 tumors/lung; 5 lungs/group). Right panels, representative micrographs

showing phosphorylated histone 3 staining. (E) Left panels, the percentage of tumors with high and

low staining for phosphorylated ERK1/2 (pERK). Right panels, representative micrographs showing

pERK staining. Scale bars, 100 µm. * P < 0.05; ** P < 0.01; *** P < 0.001. Exact P values are

provided in Table S2. Data on graphs are presented as mean ± SEM.

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Fig. S4. Antioxidants do not affect the amounts of apoptotic and senescent cells in tumors of mice

with K-RASG12D– and B-RAFV600E–induced lung cancer. (A) Immunofluorescence images of

TUNEL-stained lung sections. Positive control, thymus. (B–D) Images of lung sections stained with

antibodies recognizing cleaved caspase 3 (positive control, thymus) (B), p16 (C), and p21 (D). (E)

Levels of p19 in K-RASG12D tumors were essentially undetectable. Positive control, lung tumor

from a K-RASG12DTrp53∆/∆ mouse. Basal levels of p19 were detectable in B-RAFV600E tumors and

reduced in response to antioxidants. Similar results were observed in lungs of at least 5 mice per

genotype and treatment. Scale bars, 100 µm. Exact P value is provided in Table S2.

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Fig. S5. NAC and Trolox increase the proliferation of fibroblasts expressing oncogenic, but not wild-

type, K-RAS and B-RAF. (A) Proliferation of primary K-RASLSL/+ and B-RAFCA/+ fibroblasts in

medium supplemented with 250 µM (low) and 1 mM (high) NAC or 25 µM (low) and 100 µM (high)

Trolox. Values are the mean proliferation of fibroblasts from three embryos per genotype assayed in

triplicate. The cells were the same as in Fig. 3A but were infected with a control βgal-adenovirus.

(B–D) Cells from experiment in Figure 3A. (B) Quantification of BrdU incorporation. (C)

Quantification of cells in the S phase of the cell cycle. (D) Quantification of Annexin V-positive

cells. * P < 0.05. Exact P values are provided in Table S2. Data on graphs are presented as mean ±

SEM.

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Fig. S6. Antioxidants increase the proliferation of MYC-transformed fibroblasts. (A) Western blot

showing increased expression of c-MYC in lysates of primary fibroblasts transfected with a

retrovirus encoding c-MYC. Actin was the loading control. (B) TaqMan analyses showing increased

c-MYC expression in transfected cells. (C) Proliferation of wild-type c-MYC-transfected primary

fibroblasts. The medium was supplemented with 1 mM NAC or 100 µM Trolox. (D) Proliferation of

c-MYC-transfected Trp53-deficient primary fibroblasts in medium supplemented with NAC or

Trolox. For C and D, values are the mean proliferation of fibroblasts from three embryos assayed in

duplicate. *** P < 0.001. Exact P value is provided in Table S2. Data on graphs are presented as

mean ± SEM.

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Fig. S7. Antioxidants increase the proliferation of human lung cancer cell lines expressing wild-type

p53. (A) Quantification of BrdU incorporation in human lung cancer cell lines cultured in medium

supplemented with 1 mM NAC or 100 µM Trolox. Values are the mean of triplicate analyses per cell

line. (B) Quantification of cells in the S phase of the cell cycle. (C) Quantification of Annexin V-

positive cells. * P < 0.05. Exact P values are provided in Table S2. Data on graphs are presented as

mean ± SEM.

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Fig. S8. TP53 is required for antioxidants to increase the proliferation of human lung cancer cell

lines. (A) Western blots of cell lysates showing levels of p53 in cell lines incubated with lentiviruses

expressing shRNAs targeting TP53 or containing a scrambled (SCR) sequence. β-tubulin was the

loading control. (B) Quantification of BrdU incorporation of cells from Figure 4F. Values are the

mean of triplicate analyses per cell line. * P < 0.05. Exact P values are provided in Table S2. Data on

graphs are presented as mean ± SEM.