SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION doi: /nature12138 Supplementary Figure 1. Knockdown of KRAS leads to a reduction in macropinocytosis. (a) KRAS knockdown in MIA PaCa-2 cells expressing KRASspecific shrnas (KRAS sh 1 and KRAS sh 2) was confirmed by immunoblotting with KRAS-specific antibodies. MIA PaCa-2 cells were also transduced with a control scramble shrna. As a loading control for the Western blot, the levels of -tubulin are shown. (b) Knockdown of oncogenic KRAS results in reduced proliferation, but is tolerated in two dimensions. Relative cell number was determined by Syto 60 staining and quantified at the indicated intervals. (c and d) FITC-dextran (green) uptake was reduced in cells expressing KRAS-specific shrnas, relative to control cells expressing a scramble shrna. A comparable level of expression between the various shrnas was ensured by monitoring RFP intensity (red), which is concomitantly expressed with each shrna. The extent of macropinocytosis was quantified as described in Fig. 1 and data are presented relative to the values obtained for the scramble control cells. For image-based quantification, at least 200 cells were scored per experiment. Error bars indicate mean +/- s.e.m. for n=3 independent experiments. Statistical significance was determined via Student s t-test; ***P <

2 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 2. Oncogenic HRAS-expressing bladder cancer cells display elevated levels of macropinocytosis. (a) A macropinocytosis uptake assay utilizing TMR-dextran as a marker of macropinosomes (red) indicates that T24 cells display elevated levels of macropinocytosis compared to 5637 cells. DAPI staining (blue) identifies nuclei. Quantification of macropinocytic uptake was performed as described in Fig. 1. Data are expressed as arbitrary units and are presented relative to the values obtained for 5637 cells. (b) Macropinocytic uptake in T24 cells is inhibited by treatment with EIPA (75 M). Quantification of macropinocytic uptake was determined at the indicated concentrations of EIPA. Data are presented relative to the values obtained for the 75 M condition. (c) Relative levels of macropinocytic uptake in MIA PaCa-2, T24, BxPc-3 and 5637 cells. For all graphs, error bars indicate mean +/- s.e.m. for n=3 independent experiments with at least 300 cells scored per experiment. Statistical significance was determined via Student s t-test; **P <

3 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 3. Macropinocytosis in an autochthonous mouse model of pancreatic cancer. Representative images of pancreatic tissue obtained from P48-cre; lsl-kras G12D ; Trp53 -/+ mice that were injected with FITC-dextran (green) are shown. Sections were counterstained with anti-ck19 (red), which labels the epithelial cells in the pancreatic intraepithelial neoplasia lesions. The left-most image represents a higher magnification of the indicated boxed region. 3

4 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 4. Macropinocytic stimulation in cells harboring endogenous oncogenic Ras mutations mediates the internalization of extracellular albumin. FITC-BSA (green) is internalized into discrete puncta that colocalize with TMR-dextran (red) in MIA PaCa-2 (top panel) and T24 (bottom panel) cells. DAPI staining (blue) identifies nuclei. Images shown are representative of at least n=3 independent experiments. 4

5 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 5. Macropinocytosed albumin is targeted for proteolytic degradation in cancer cells harboring oncogenic Ras mutations. (a, b) Analysis of DQ-BSA fluorescence in MIA PaCa-2 (a) and T24 (b) cells that were co-incubated with DQ-BSA (green) and TMR-dextran (red) and fixed either immediately at the end of the incubation (t = 0) or following a 1-hour chase in media free of both DQ-BSA and TMRdextran (t = 1 h). The fluorescent signal emanating from DQ-BSA is an indication of albumin degradation. Insets in (b) represent a higher magnification of the boxed region. All images shown are representative of at least n=3 independent experiments. 5

6 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 6. Degradation of DQ-BSA in oncogenic Ras-expressing cells is blocked by bafilomycin A1, an inhibitor of lysosomal hydrolase activity. Analysis of DQ-BSA fluorescence in MIA PaCa-2 (top panel) and NIH KRas V12 (bottom panel) cells indicates that treatment with bafilomycin A1 blocks DQ-BSA degradation. Cells were co-incubated with DQ-BSA (green) and TMR-dextran (red) for minutes and subsequently chased for 1 hour in media containing DMSO (vehicle control) or 100 nm bafilomycin A1. All images shown are representative of at least n=3 independent experiments. 6

7 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 7. Culturing of NIH 3T3 KRas V12 cells in physiological levels of albumin increases intracellular levels of glutamate and -ketoglutarate. Intracellular levels of glutamate and -ketoglutarate are elevated following the incubation of NIH 3T3 KRas V12 cells in complete medium supplemented with 2% albumin (CM+Alb) compared to complete medium alone (CM). Intracellular accumulation of both metabolites was blocked by 25 M EIPA treatment (CM+Alb+EIPA). Data are expressed as arbitrary units and are presented relative to the values obtained for the CM condition. Error bars indicate mean +/- s.e.m. for n=3 independent experiments. Statistical significance was determined via Student s t-test; *P < 0.05, ***P <

8 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 8. Amino acid labeling in culture media with 13 C-labeled yeast protein. (a) Amino acid pools in fresh (t = 0) medium contains almost exclusively unlabeled amino acids. (b) Minimal 13 C-labeling in amino acids is seen when culture medium containing 13 C-labeled protein is incubated for 24 hours without cells, however the same media incubated for the same time period with NIH 3T3 KRas V12 cells contains significant amounts of fully 13 C-labeled amino acids. Error bars indicate mean +/- s.d. for n=3 independent experiments. Some amino acids were omitted due to low signal or their absence in DMEM. 8

9 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 9. Model depicting the metabolism of protein-derived serine and glycine. Intracellular serine and glycine pools contain significant 13 C- labeling. Partially labeled isotopomers likely arise due to bidirectional activity of serine hydroxymethyltransferase (SHMT). Error bars indicate mean +/- s.d. for n=3 independent experiments. M0 corresponds to metabolites with no label, M1 with one 13 C label, M2 with two 13 C labels, and M3 with three 13 C labels. 9

10 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 10. The adverse effects of glutamine deprivation on the proliferation of various oncogene-transformed cell lines. MIA PaCa-2, T24, NIH 3T3 KRas V12 and NIH 3T3 Src Y527F cells were cultured at the indicated concentrations of glutamine and total viable cell counts were measured by MTT assay after 0, 2 and 4 days of growth. All three cell lines exhibit compromised cell growth at sub-physiological concentrations of glutamine compared to physiological glutamine levels (0.5 mm). Concentrations shown represent the lowest concentration at which cell proliferation was suppressed without affecting viability. Data are presented relative to the values obtained at Day 0. Error bars indicate mean +/- s.e.m. for n=3 independent experiments. 10

11 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 11. Extracellular albumin rescues the proliferative effects of glutamine deprivation as quantified via Syto 60 staining. Relative cell number was quantified via a Syto 60 assay, which is independent of mitochondrial activity. Assessment of proliferative capacity, under the indicated conditions via Syto 60 staining, yielded results similar to those obtained by MTT quantification. The compromised proliferation of oncogenic Ras-expressing cells resulting from growth in media containing sub-physiological concentrations of glutamine (0.2 mm, 0.2Q) is reversed by supplementation with 2% albumin (0.2Q+Alb) and this effect is inhibited by treatment with 25 M EIPA (0.2Q+Alb+EIPA). Cell number was determined after 6 days of growth and data are presented relative to the values obtained for the 0.2Q condition. A representative image of Syto 60-stained MIA PaCa-2 cells grown under the indicated conditions is shown on the left. Error bars indicate mean +/- s.e.m. for n=3 independent experiments. Statistical significance was determined via Student s t-test; *P < 0.05, ***P <

12 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 12. The ability of extracellular albumin to rescue the proliferative defects caused by glutamine deprivation is dependent on oncogenic KRAS expression. MIA PaCa-2 cells expressing control scramble shrna or KRASspecific shrnas (KRAS sh 1 and KRAS sh2) were grown in media containing subphysiological concentrations of glutamine (0.2 mm, 0.2Q) with and without albumin supplementation. Total viable cell counts were measured after 6 days of growth and data are presented relative to the values obtained for the 0.2Q condition. Error bars indicate mean +/- s.e.m. for n=3 independent experiments. Statistical significance was determined via Student s t-test; ***P <

13 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 13. Src Y527F -induced macropinosomes facilitate the internalization and degradation of extracellular albumin and the decrease in proliferation of Src-transformed cells following glutamine deprivation is rescued by albumin supplementation. (a) TMR-dextran (red) is internalized at significantly higher levels in NIH 3T3 Src Y527F (Src Y527F ) cells compared to untransformed control cells (CTL). Quantification of macropinocytic uptake was performed as described in Fig. 1 and data are presented relative to the values obtained for the untransformed control cells. Error bars indicate mean +/- s.e.m. for n=3 independent experiments with at least 125 cells scored per experiment. (b) Analysis of DQ-BSA fluorescence in NIH 3T3 Src Y527F cells that were co-incubated with DQ-BSA (green) and TMR-dextran (red) and fixed either immediately at the end of the incubation period (t = 0) or following a 1-hour chase in media free of both DQ-BSA and TMR-dextran (t = 1 h). The fluorescent signal emanating from DQ-BSA is an indication of albumin degradation. Macropinosomes containing degraded DQ-BSA (yellow in merged image) are indicated by the arrowheads. (c) The reduced proliferative capacity of Src Y527F -expressing cells resulting from growth in media containing sub-physiological concentrations of glutamine (0.1 mm, 0.1Q) is reversed by supplementation with 2% albumin (0.1Q+Alb). Total viable cell counts were measured after 6 days of growth and data are presented relative to the values obtained for the 0.1Q condition. Images shown in (a) and (b) are representative of at least n=3 independent experiments. Statistical significance was determined via Student s t-test; ***P <

14 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 1. Amino acid fragments used for isotope quantification. Metabolite Carbons Formula m/z range Ala 123 C11H26O2NSi Gly 12 C10H24O2NSi Val 1234 C13H30O2NSi Leu C13H32ONSi Ile C13H32ONSi Ser 123 C17H40O3NSi Glu C19H42O4NSi