Supplementaryfigure Legends

SupplementaryFigure Legends

Supplementary Figure S1.

Analysis of G6PD mRNA expression. Reverse transcription (RT) and quantitative real-time PCR (qPCR) were performed to determine G6PD mRNA levels in seven human breast cancer cell lines following exposure to 20% or 1% O2 for 24 hours. For each sample, the expression of each mRNA was quantified relative to 18S rRNA and then normalized to the result obtained from MDA-MB-231cells (MDA-231) at 20%O2 (mean ± SEM; n = 3). Expression levels at 1% O2that are significantly decreased compared with 20% O2are shown as black bars(P < 0.001; Student’s t test).

Supplementary Figure S2.

Analysis of MCF-7 Subclones.A,immunoblot assays were performed to analyze HIF-1α, PHGDH, PSAT1, and Actin protein expression in whole cell lysates prepared from MCF-7subclones, which were stably transfected with lentiviral vectors encoding a non-targeting control shRNA (NTC) or shRNA targeting HIF-1 (sh1), HIF-2 (sh2), or HIF-1 and HIF-2(double knockdown [DKD]), and exposed to 20% or 1% O2 for 48 hours. B,immunoblot assays were performed to analyzeHIF-1α, PHGDH, PSAT1, PSPH and Actin protein expression using whole cell lysates prepared from MCF-7 cells treated with vehicle control (0) or acriflavine (Acr; 2.5 or 5 µM) and exposed to 20% or 1% O2 for 48 hours. C, MCF-7 subcloneswere exposed to 20% or 1% O2 for 72 hours andthe percentage of aldehyde dehydrogenase-expressing (ALDH+) cells was determined byAldefluor staining and flow cytometry (mean ± SEM; n = 3). *P < 0.01 vs 20% O2; #P < 0.01 vs 1% O2(ANOVA). D, parental MCF-7 cells were treated with vehicle (Con) oracriflavine (Acr) and exposed to 20% or 1% O2 for 72 hours. Cells were transferred to ultra-low attachment plates, and 7 days later the number of primary mammospheres per field was counted (mean ± SEM; n = 3). *P0.001 vs.20% O2/0 Acr; #P < 0.001 vs 1% O2/0 Acr (ANOVA). Representative photomicrographs of mammospheres are shown (left; scale bar, 2 mm). E,MCF-7 cells were cultured on standard tissue culture plates as adherent monolayer cultures or on ultra-low adherence plates for 7 days to generate primary mammospheres. The adherent cells and mammospheres were harvested for RT-qPCR analyses. Results were normalized to adherent cells for each mRNA (mean ± SEM; n = 3). *P0.001 vs.adherent cells (Student’s t test).

Supplementary Figure S3.

Effect of PHGDH knockdown on cell growth. A and B,MDA-MB-231 (A) and MCF-7 (B) subclones, which were stably transfected with a lentiviral vector encoding a non-targeting control shRNA (NTC) or any one offive different shRNAs targeting PHGDH (sh1, sh2, sh3, sh4, sh5), were exposed to 20% or 1% O2 for 72 hours. Cell viability was measured by MTT assay and, for each cell line, normalized to the NTC subclone at 20% O2 (mean + SEM; n = 5). *P < 0.01 vs. NTC at 20%O2; #P< 0.01 vs. NTC at 1%O2 (ANOVA).

Supplementary Figure S4.

Effect of carboplatin on cell viability.MCF-7 (A) and MDA-MB-231 (B) subclones were exposed to increasing concentrations of carboplatin for 72 hours under 20% O2 and cell viability was determined by MTT assay. Viability was expressed relative to vehicle-treated cells.

Supplementary Figure S5.

Effect of doxorubicin treatment. MDA-MB-231 (left) and MCF-7 (right) cells were exposed to doxorubicin at IC50 for 72 hours and analyzed for MitoSox+ (A), apoptotic (B), and ALDH+ (C) cells by flow cytometry. *P < 0.01 vs. NTC at 20% O2; #P< 0.01 vs. NTC at 1% O2 (ANOVA).

Supplementary Figure S6.

Clinical correlates.A, S1Csignature is significantly associated with tumor gradeby ANOVA in an analysis of 1,160 breast cancers using the GOBO database.B,Kaplan–Meier analyses of relapse-free survival over 120 months were performed based on clinical and molecular data from 3,554 breast cancer patients, who were stratified by expression of PSAT1 (upper left), PSPH (upper right), SHMT2 (lower left), or MTHFD2 (lower right)mRNA in the primary tumor, which was greater (red) or less (black) than the median. TheP value (log-rank test) and hazard ratio (HR) for each comparison are shown.