A. Generation and characterization of Ras-expressing autophagycompetent

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1 Supplemental Material Supplemental Figure Legends Fig. S1 A. Generation and characterization of Ras-expressing autophagycompetent and -deficient cell lines. HA-tagged H-ras V12 was stably expressed in autophagy-competent or autophagy-deficient ibmk cells and the protein levels of HA-H-ras, Atg5-Atg12, Beclin1, and p62 were analyzed by Western blotting. Note that Western blotting for Atg5 detects the Atg5-Atg12 conjugation product that is absent from both atg5 -/- and atg7 -/- cells and that allelic loss of beclin1 results in an approximate 50% reduction in Beclin1 protein expression. Levels of the autophagy substrate p62 are highest with complete autophagy deficiency. B. Stable expression of H-ras V12 elevates basal autophagy and allelic loss of beclin1 produces a partial autophagy defect. Autophagy-competent (beclin1 +/+ ) or -deficient cells (beclin1 +/- ) expressing H-ras V12 were transiently transfected with the fluorescent autophagosome marker p-tfl-lc3 and allowed to recover overnight prior to starvation. Cells were treated with 1nM bafilomycin A1 and incubated with HBSS for the indicated periods. Representative images depict LC3 localization. Numbers in upper left of each panel indicate percentage of cells with LC3 translocation to autophagosomes (punctate localization). Bar represents 10µm. C. Allelic loss of beclin1 and a partial defect in autophagy does not impair processing of endogenous LC3-I to LC3-II. beclin1 +/+ and beclin1 +/- ibmk cells expressing H-ras V12 were treated with HBSS. Cells were then collected at the

2 indicted time points and analyzed for active caspase-3 and LC3 I cleavage to LC3 II that is autophagosome associated. D. Quantification of RFP-LC3 puncta-positive cells of Fig.1A. Graph bars show the percentage of RFP-LC3 puncta-positive cells. Error bars represent SD from two independent experiments (asterisks show a significant difference as compared to atg5+/+-pcgn control by repeated measure ANOVA Bonferroni posttest: p<0.05). E. mtor activation is maintained in Ras-expressing cells with high basal autophagy. Autophagy-competent or -deficient ibmk cells expressing H-ras V12 or the pcgn vector control under nutrient replete conditions were assessed for phosphorylation of the mtor substrate S6 by Western blotting. Atg5+/+pCGN cells treated with HBSS for 3hr to inhibit mtor were used for negative control for S6 phosphorylation. F. Quantification of clonogenic survival from Fig. 1D. Bar Graphs show the survival fraction after HBSS starvation treatment normalized to non-treatment. Survival fraction was quantified by cell density using ImageJ software. G and H, Partial autophagy defect does not impair viability or clonogenic survival in starvation. G. beclin1 +/+ and beclin1 +/- ibmk cells expressing H- ras V12 cells were treated with HBSS for 22hrs and cell viability was examined. H, Cells treated as in (G) were allowed to recover in normal medium for 2 days and then stained with Giemsa to assess clonogenic survival.

3 I. Staurosporine induces cell death of both atg5 +/+ and atg5 -/- cells expressing H-ras V12. atg5 +/+ or atg5 -/- ibmk cells expressing H-ras V12 were treated with 1µM staurosporine for 24hrs. Cell viability was analyzed by trypan blue exclusion and normalized with untreated cells at the time of initiation of starvation. J. Methyl-pyruvate (MP) treatment does not rescue HBSS starvationinduced atg5 -/- H-ras V12 ibmk cell death. atg5 +/+ H-ras V12 or atg5 -/- H-ras V12 cells were treated with HBSS without or with methyl-pyruvate (0.5mM) for 12hrs. Cell viability was analyzed by trypan blue staining and normalized to untreated cells at the time of initiation of starvation. K and L. Starvation-induced cell death is not caused by ROS. K, The ROS scavenger N-Acetyl-L-cysteine (NAC) does not rescue HBSS starvation-induced atg5 -/- H-ras V12 ibmk cell death. atg5 +/+ H-ras V12 or atg5 -/- H-ras V12 ibmk cells were treated with HBSS without and with NAC (1.5mM) for 12hrs, and cell viability was determined by trypan blue staining and normalized to the time of initiation of starvation. L, Overlays showing ROS levels in Ras-expressing, autophagycompetent and -defective ibmk cells under normal (blue) and 3hrs starvation (HBSS) (red) conditions, by flow-cytometry using the ROS sensor dichlorodihydrofluorescene diacetate (DCF-DA). Numbers indicate mean fluorescence signal for 10 4 cells. M. Autophagy or p62 deficiency does not affect H-ras V12 activity. The GST- Raf1-RBD pull down assay was performed in the cell lysates from atg7 +/+ H-

4 ras V12, atg7 -/- H-ras V12, p62 +/+ H-ras V12 or p62 -/- H-ras V12 ibmk cells. Active GTPbound H-ras V12 levels were determined by Western blot with anti-ras antibody. Fig. S2. A. Generation and characterization of K-ras V12 -expressing Atg5-wild type and -deficient ibmk cell lines. HA-tagged K-ras V12 was stably expressed in atg5 +/+ or atg5 -/- ibmk cells and the protein levels of HA-H-ras, Atg5, and p62 were analyzed by Western blotting. B. Stable expression of K-ras V12 elevates basal autophagy. Autophagycompetent (atg5 +/+ ) or -deficient cells (atg5 -/- ) expressing K-ras V12 were transiently transfected with the fluorescent autophagosome marker p-tfl-lc3 and allowed to recover overnight prior to starvation. Cells were treated with 1nM bafilomycin A1 and incubated with HBSS for the indicated periods. Representative images depict LC3 localization. Numbers in upper left of each panel indicate percentage of cells with LC3 translocation to autophagosomes (punctate localization). Bar represents 10µm. C. Evaluation for processing of endogenous LC3-I to LC3-II and activation of caspase-3 in K-ras V12 expressing ibmk cells. Atg5 +/+ and atg5 -/- ibmk cells expressing K-ras V12 were treated with HBSS. Cells were then collected at the indicted time points and analyzed for active caspase-3 and LC3 I cleavage to LC3 II that is autophagosome associated. D and E, Autophagy deficiency impairs viability and clonogenic survival in starvation. D. atg5 +/+ and atg5 -/- ibmk cells expressing K-ras V12 cells were treated with HBSS for 8hrs and cell viability was examined. E, Cells treated as in

5 (D) were allowed to recover in normal medium for 2 days and then stained with Giemsa to assess clonogenic survival. F, G and H, autophagy deficiency impairs K-ras V12 tumorigenesis. F. Tumor growth of atg5 +/+ and atg5 -/- cells expressing K-ras V12. G, Representative images of tumor bearing mice at day 15 post-injection are shown. H, Representative images of paraffin-embedded tumor sections stained by H&E, and IHC for active caspase-3, p62 or Ub in tumors from (A). Error bars represent standard errors. *P<0.05, ** P<0.01 (T-test). Fig. S3 A, B and C. Allelic loss of beclin1 has modest impact on H-ras V12 tumorigenesis. A. Tumor growth of beclin1 +/+ and beclin1 +/- cells expressing H- ras V12. Error bars represent standard errors. *P<0.05 (T-test). B, Representative images of tumor bearing mice at day 15 post-injection are shown. C, Representative images of paraffin-embedded tumor sections stained by H&E, and IHC for active caspase-3, p62 or Ub in tumors from (A). D. Autophagy deficiency prevents LC3 translocation in tumors. p-tfl-lc3 tandem-tagged LC3-expressing atg5 +/+ H-ras V12 or atg5 -/- H-ras V12 ibmk cells were subcutaneously injected into nude mice. Frozen sections of tumors at day 8 post-injection were imaged by confocal microscopy. Representative images show LC3 translocation (red) in tumors in atg5 +/+ H-ras V12 (indicated by an arrow), but not atg5 -/- H-ras V12 tumors. DAPI was used to stain nuclei (blue). Fig. S4

6 A. Quantification of clonogenic survival from Fig. 3C. Bar Graphs show the survival fraction after HBSS starvation treatment normalized to non-treatment. Survival fraction was quantified by cell density using ImageJ software. Fig. S5 A, EMs of tumors from beclin +/+ H-ras V12 or beclin1 +/- H-ras V12 expressing ibmk cells. Representative EMs show the accumulation of abnormal mitochondria in the tumor from compared to the tumor from beclin1 +/+ ibmk cells expressing H-ras V12. B and C, Autophagy deficiency impairs starvation-induced mitophagy. B, Starvation (HBSS) promotes mcherry-parkin mitochondrial translocation. H- ras V12 expressing atg5 +/+ or atg5 -/- ibmk cells stably expressing mcherry-parkin (red) were treated with HBSS or 20µM CCCP for 4hrs. Representative images are shown for each time point. Numbers in upper left of each panel indicate the percentage of cells with mcherry-parkin, which has translocated to mitochondria. Bar represents 10µm. C, Autophagy-competent or -deficient ibmk cells expressing H-ras V12 were treated with HBSS and samples were collected at the indicated time points, then Western blotted with anti-tom20. D, Autophagy-deficient cells are more sensitive to loss of mitochondrial membrane potential in starvation. Autophagy-competent or -deficient ibmk cells expressing H-ras V12 or vector control pcgn were treated with HBSS at the indicated time point and then stained with Δψm-dependent MitoTracker-Red (to

7 monitor mitochondria membrane potential change) and MitoTracker-Green (to indicate total mitochondria). Cells were analyzed by flow cytometry. Fig. S6 A. Quantification of clonogenic survival from Fig. 5C. Bar Graphs show the survival fraction of different treatment (DMEM+DMSO/ CCCP; HBSS+DMSO/CCCP) normalized to DMEM+DMSO treatment. Survival fraction was quantified by cell density using ImageJ software. B. Pool sizes of major TCA cycle intermediates in atg5 +/+ -Bcl-2 and atg5 -/- - Bcl-2 cells under nutrient replete and starvation conditions. Graphs showing cell number normalized relative pool sizes (a.u.) of major TCA metabolites by LC-MS in HBSS for indicated times. C. Allelic loss of beclin1 impairs mitochondrial respiration capacity. Representative graphs show oxygen consumption rates in Ras-expressing, autophagy-competent (beclin1 +/+ ) and -deficient cells (beclin1 +/- ) ibmk cells under normal, and following addition of FCCP (1.5µM) and the complex III inhibitor antimycin (20µM) as in Fig. 5E. The beclin1 +/+ Hras2-15 and beclin1 +/- Hras2 cell lines with higher Ras expression and dependency on autophagy for tumorigenesis (Fig. S3A, B) are shown. D. Ras expression causes energy depletion in autophagy defective cells under starvation. Bar graphs show ATP/ADP ratio (upper panel) and AMP levels (nmols/ml) (lower panel) in atg5 +/+ and atg5 -/- ibmk cells without and with H-ras V12 expression under normal and starvation (HBSS) conditions. Data points

8 show mean ± SD. Statistical significance was calculated by 2-way ANOVA with Bonferroni posttest.

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