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1 doi:.38/nture73 Glucose SSP THF Methyl- THF Purines 3- PG PHGDH PSAT PSPH Glycine SHMT GSH Lctte Pyruvte ROS TCA Cycle Glucose p53 p Purines 3- PG PHGDH PSAT PSPH Glycine SHMT GSH Lctte Pyruvte ROS TCA Cycle Supplementry Figure. Removl of exogenous serine cuses energe@c stress nd p53- dependent metolic remodeling in cncer cells. Cncer cells rpidly udlise exogenous serine, which is converted within cells to glycine, used to synthesise purine nucleoddes, GSH nd other metolites. Conversion of serine to glycine lso provides methyl- THF, n importnt intermedite in the synthesis of purine nd pyrimidine nucleoddes, wheres the reverse (conversion of glycine to serine) consumes methyl- THF. The serine synthesis pthwy (SSP) udlises the glycolydc intermedite 3- PG, which is converted y PHGDH, PSAT nd PSPH into serine. Removl of exogenous serine cuses cdvdon of the SSP, ccompnied y decresed flux to lctte, resuldng in ATP depledon. To compenste, more pyruvte is trnsferred to the TCA cycle for elevted OXPHOS. p53- dependent cdvdon of p induces trnsient cell cycle rrest, locking flux to purines, thus mintining GSH synthesis. Enzymes shown in old typefce, metolites in norml typefce. 3- PG; 3- phosphoglycerte, GSH; glutthione, ROS; recdve oxygen species, THF; tetrhydrofolte.
2 RESEARCH SUPPLEMENTARY INFORMATION RKO RKO p53- /- RelDve cell numer Dys % Su- G cells Gly - Ser c % PI+ (ded) cells 3 - Gly - Ser wt Null p53- /- p53- /- d 8 MEF 5 MEF p53- /- RelDve cell numer Dys e RelDve cell numer h h h h HCT6 5 5 HCT6 p53- /- (ex) + Lysine - Lysine Dys f % Glycine in medi p53- /- (ex) Supplementry Figure. p53 supports prolifer@on of RKO cells nd MEFs during serine strv@on., RKO cells were grown in complete medi or medi deficient in serine nd glycine (- SG)., cell deth in RKO cells ws ssessed y PI stining of fixed cells (n=5) nd c, PI exclusion y live cells (n=3), oth qundfied y flow cytometry. d, MEFs were grown in complete medi or medi deficient in serine nd glycine (- SG). e, HCT6 cells were grown in cell culture medis formulted with individul nutrient components to pproximtely mtch DMEM, either lcking (- ) or contining (+) the essendl mino cid lysine. f, HCT6 cells were grown in complete medi, glycine levels present in the spent medi were qundfied y LC- MS (verges of triplicte wells vs. fresh medi). Cell proliferdon curves re verges of triplicte wells. All error rs re SEM.
3 RESEARCH 35 Averge weight (g) Control Dys cer injecdon 5.E+65.E+6 3.E+63.E+6.E+6.E+ Are x 6 Glycine Alnine Arginine Asprtte.E+6.6.E+7. 8.E+68.E+6. Are x 6 * 8.E+5.8 *.E+5..E+7.5 Are x 7.E+7. 5.E E+66.E+6.E+6.E+.E+.E+.E+6 8.E E+5 6.E+5.E+5.E+ Are x 6 Are x 5 Asprgine Glutmte Glutmine HisDdine Proline.E+6..E+6..E+8. 3.E E+7 6.E+6.5.E+7. 8.E E+7.8.E+7.E+7.5.E+6..E+5..E+7..E+7..E+7 5.E E+6.5.E+.E+.E+.E+.E+ Control - SG Are x 6 Are x 6 Are x 8 Are x 7 Are x 7 Tyrosine Isoleucine Leucine Lysine Methionine.E+7. 3.E+7 3.E+7 8.E+68 3.E+7 3.E+7.5.E+7 3.E+73 6.E+66.E+7.E+7..E+7.E+6 5.E+6.5.E+7.E+7.E+6.E+7.E+.E+.E+.E+.E+ Control - Ser - SG& Gly Control - Ser - SG& Gly Control - SG Are x 7 Are x 7 Are x 7 Are x 6 Are x 7 Phenyllnine Threonine Tryptophn Vline 3.E E E E+7 5.E+7..E+7.E+7.E+7.5.E+7 3.E+7 3.E+7..E+7.E+7.E+7 5.E+6.5.E+7.E+.E+.E+.E+ Control - SG Are x 7 Are x 7 Are x 7 Are x 7 Supplementry Figure 3. The effects of serine nd glycine deficient diet in vivo., The weights of mice used in the xenogrc experiment were recorded t regulr intervls. Wstge of food in some cges (Control nd Ser & Gly diets) mde it difficult to ccurtely record diet consumpdon. In cge where wstge did not occur, consumpdon of Ser & Gly diet ws 6g/dy/mouse, comprle to the predicted norml intke of 5g/dy/mouse., LC- MS ws performed on serum smples tken from the mice t Dme of scrifice to evlute mino cid content (*p<.5). Control diet n=, - SG diet n=8, ll error rs re SEM. 3
4 RESEARCH SUPPLEMENTARY INFORMATION p53- /- (ex) h h h h h h PHGDH PSAT PSPH PGAM CDK PHGDH PSAT PSPH PGAM CDK Intensity vs. h 3 PHGDH / CDK 8 6 PSPH / CDK h h h h h h h h 6 PSAT / CDK p53- /- (ex) PGAM / CDK Intensity vs. h h h h h h h h h Supplementry Figure. nd glycine strv@on results in up- regul@on of SSP enzymes., RepresentDve western lots for PHGDH, PSAT, PSPH nd PGAM protein expression in HCT6 cells either fed complete or serine nd glycine deficient (- Ser & Gly) medi., Protein expression ws qundfied vi infrred tgged secondry ndodies on LiCor infrred scnner. Expression ws normlised to CDK expression. Dt is the verge of three independent experiments, error rs re SEM.
5 RESEARCH Phos- p85s6k (Thr) Phos- p7s6k (Thr389) p53- /- (ex) 8h 8h 8h 8h p85s6k p7s6k Phos- S6 (Ser/) S6 Supplementry Figure 5. mtorc is modulted in p53- independent mnner during serine HCT6 cells were grown t low density in complete () or serine nd glycine deficient (- SG) medi. A western lot ws proed for mrkers of mtorc cdvity p7s6k nd S6 re downstrem of mtorc nd re cdvted y phosphoryldon when mtorc is cdve in response to growth promodng signls. 5
6 RESEARCH SUPPLEMENTARY INFORMATION Leled (see individul metolites) Hexose phosphte M+6 Unleled M+.E+6.5 Are x 6.E+6. 5.E+5.5.E+ 5 SG SG h SG h 5 - h - 5 P h P h + Pyr P 5 h h 5 h h 5 h h Glycerldehyde 3- phosphte M+6.E+5 3.E+53.E+5.E+5.E+ 5 SG h SG h SG 5 - h - 5 P h P h P 5 h h 5 h h - 5 SG h + Pyr h Are x 5.E+5 3.E+53.E+5.E+5.E+ Are x 5 M+3 5 SG SG h SG h 5 - h - 5 P h P h P 5 h h 5 h 5 SG h + Pyr h M+3 3.E+7 3 Are x 7.E+7.E+7.E+ Lctte M+3 5 SG SG h SG h 5 - h - 5 P h P h P + Pyr 5 h h 5 h h 5 h h.e+7.5 Are x 7.E+7. 5.E+6.5.E+ Pyruvte M+3 5 SG SG h SG h 5 - h - 5 P h P h P + Pyr 5 h h 5 h h 5 h h.e+7.5 Are x 7.E+7. 5.E+6.5.E+ 5 SG h SG h SG 5 - h - 5 P h P h P 5 h h 5 h 5 SG h + Pyr h Citrte / Isocitrte M+ Cis- Aconitte M+.E+6.5 Are x 6.E+6. 5.E+5.5.E+ 5 h h 5 h h 5 h h SG SG SG P P P + Pyr 5 h h 5 h h 5 h h.e+6.5 Are x 6.E+6. 5.E+5.5.E+ 5 h h 5 h h 5 h h SG 5 SG h SG h h - h P 5 P h P h + Pyr Supplementry Figure 6. Pyruvte pr@lly rescues to growth of p53- /- cells during serine nd glycine strv@on y ugmen@ng TCA cycle flux. HCT6 p53- /- (ex) cells were grown in complete medi or serine nd glycine deficient medi (- SG), or serine nd glycine deficient medi supplemented with 5mM pyruvte (- SG + Pyr). Acer, medi ws replced with equivlent medi contining U- 3 C- leled D- Glucose for the indicted Dmes. The reldve qunddes of intrcellulr metolites were nlysed y LC- MS. Dt is verge of triplicte wells, error rs re SEM. 6
7 RESEARCH p53 p CDK Control nm nm 3nM Intensity vs. Control 3 Control nm nm 3nM Control nm nm 3nM p53 / CDK p / CDK MPA MPA c Phos- AMPK (Thr7) AMPK CDK h h Supplementry Figure 7. Inhii@on of GMP synthesis replictes the p53- p response induced y serine strv@on., HCT6 cells were treted with mycophenolic cid (MPA) for hours. A western lot ws proed for p53 nd p expression., Protein expression ws qundfied vi infrred tgged secondry ndodies on LiCor infrred scnner nd normlised to CDK expression. c, HCT6 cells were grown in complete medi or serine nd glycine deficient medi (- Ser & Gly). A western lot ws proed for AMPK cdvity. 7
8 RESEARCH SUPPLEMENTARY INFORMATION 8.E+8 AMP Are x E+6.E+6.E+6.E+ p53- /- (ex) 5.E+35.E+3 Are x 3 3.E+33.E+3.E+3.E+ IMP p53- /- (ex) 6.E+6 Are x.e+.e+.e+.e+.5 p53- /- (ex) GMP Leled M+, M+, M+5 Unleled M+ Leled M+ Unleled M+ Leled M+, M+5 Unleled M+ Are x.e+. 5.E+3.5.E+ Leled M+, M+5 Unleled M+ 3 3.E+ IMP 8.E+78 GSH (totl).e+6.5 GSH (leled only) p53- /- (ex).e+ Are x.e+ 6.E+76 Are x 7.E+7.E+7.E+6. Are x 6 5.E+5.5.E+ Wt SG Wt - p null SG p null -.E+ Wt SG Wt - p null SG.E+ Wt SG Wt - p null SG p+/+ p- /- p+/+ p- /- p+/+ p- /- p null - p null - Leled M+5 M+7 Leled M+ Unleled M+ c 6.E+66.E+6 Are x 6.E+6.E+ GSH (leled only) p+/+ p- /- Leled M+ 3.E+7.5.E+7. Are x 7.E+7.5.E+7. 5.E+6.5.E+ GSH (totl) p+/+ p- /- Leled M+ Unleled M+ Supplementry Figure 8. cells inhiit flux of serine to purine synthesis Xer serine nd glycine strv@on., HCT6 cells were either fed complete medi or medi deficient in serine nd glycine (- SG) for, followed y complete medi with U- 3 C, 5 N- lelled L- for., HCT6 cells were fed complete medi, or serine nd glycine deficient medi for, followed y U- 3 C- Glucose for hour. c, HCT6 cells were fed complete medi, or serine nd glycine deficient medi for 7 hours, followed y U- 3 - C- Glucose for 3 hours. LC- MS ws used to detect reldve qunddes of intrcellulr metolites. HCT6 cells (wild- type/prentl line) re denoted s nd p+/+ in this figure. Dt re verges of triplicte wells, ll error rs re SEM. 8
9 RESEARCH p53- /- (ex) CellROX CellROX & Hoechst CellROX CellROX & Hoechst GSH Glycine 6.E+76 Are x 7.E+7.E+7.E+ 5 h h 5 h h 5 h h SG 5 SG h SG 5 - h - h GSH GSH GSH - 5 SG h + GSH h.e+7.5 Are x 7.E+7. 5.E+6.5.E+ 5 h h 5 h h 5 h h SG 5 SG h SG h h - h GSH GSH GSH - 5 SG h + GSH h.e+7 8.E+68 6.E+66.E+6.E+6.E+ Are x 6 5 h h 5 h h 5 h h SG 5 SG h SG 5 - h - h GSH GSH GSH - 5 SG + h GSH h Leled M+ Unleled M+ Leled M+3 Unleled M+ Leled M+ Unleled M+ c 5 RelDve cell numer 3 + Pyr & GSH + Pyr + GSH 3 5 Dys Supplementry Figure 9. nd glycine strv@on elevtes ROS in p53- /- cells., HCT6 cells were either fed complete medi or medi lcking serine nd glycine (- Ser & Gly) for. An oxiddon- cdvted fluorescent dye (red) nd Hoechst nucler counter- stin (lue) were dded to the medi for 3min prior to imging., HCT6 p53- /- (ex) cells were either fed complete medi, or medi lcking serine nd glycine (- SG), or medi lcking serine nd glycine supplemented with 5mM glutthione (- SG +GSH) for, cer which equivlent medi contining U- 3 C- leled D- Glucose ws dded for the stted Dmes. The reldve qunddes of intrcellulr metolites were nlysed y LC- MS. c, HCT6 cells were fed complete medi, or medi deficient in serine nd glycine supplemented with pyruvte 5mM nd / or GSH 5mM. Dt re verges of triplicte wells, ll error rs re SEM. 9
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