SUPPLEMENTARY INFORMATION AMPK mintins energy homeostsis nd survivl in cncer cells vi regulting p38/pgc-1α-medited mitochondril iogenesis Blkrishn Chue 1, Prmnnd Mlvi 1, Shivendr Vikrm Singh 1, Noshd Mohmmd 1, Benoit Viollet 2, nd Mnoj Kumr Bht 1 Ntionl Centre for Cell Science, Svitrii Phule Pune University Cmpus, Gneshkhind, Pune, Mhrshtr, Indi 411 7
Supplementry figure S1 WT-MEF -FBS 12 +Glucose - Glucose - Glu + AICAR 1 - Glu + Rp 8 6 4 2 WT-MEF + FBS 12 + Glucose - Glucose - Glu + AICAR 1 - Glu + Rp 8 6 4 2 12 1 8 6 4 2 WT-MEF +Glucose -Glucose -Glu+AICAR -Glu+Rp 12 24 36 48 6 72 84 Time (hr) c 12 1 8 6 4 2 AMPK DKO + FBS + Glucose - Glucose - Glu + AICAR - Glu + Rp 12 1 8 6 4 2 AMPK DKO - FBS + Glucose - Glucose - Glu + AICAR - Glu + Rp d 12 1 8 6 4 2 AMPK DKO-MEF +Glucose -Glucose -Glu+AICAR -Glu+Rp 12 24 36 48 6 72 84 Time (hr) Supplementry figure S1: AMPK protects cell from glucose nd FBS deprivtion induced cell deth. () WT-MEFs were cultured in 96 well pltes. Cells were grown in DMEM with or without glucose nd FBS in presence of sence of.5 mm AICAR or 2 nm rpmycin for 72h. Growth ws ssyed y using MTT ssy t indicted time point. () Time dependent growth curve of WT-MEFs cells cultured under the condition mentioned in (). (c) Cell survivl of AMPK α1/α2 doule knock out (DKO) cells cultured under condition similr to WT cells mentioned in (). (d) Time dependent growth curve of WT-MEFs cells cultured under the condition mentioned in (). All vlues re represented s men ± SD. 2
Supplementry figure S2 Glutmine Glucose No sus Ctrl sirna AMPK sirna 4 EV 35 DN-AMPK 3 25 2 15 1 5 H1299 AICAR - + + + + + + + Lctte - - + - + - + - Glutmine - - - + - + - + Ox - - - - + + - - DON - - - - - - + + c EGCG DON Oligomycin -AICAR + AICAR d Reltive intensity (% of control) 2 15 1 5 H1299 25 Ctrl-siRNA AMPK-siRNA Veh Ctrl AICAR No sus Glu Gln EGCG DON Oli 5 mm Gln Supplementry figure S2: Inhiiting lctte nd glutmine metolism in cncer cells rogte AMPK medited cell survivl. () Cell survivl in H1299-EV nd H1299-DN cells cultured in DMEM without glucose or in presence of sence of 1 mm lctte nd 5 mm glutmine. Cells were treted with 25 mm oxmte, n inhiitor of enzyme LDH, or 1 mm 6- Dizo-5-oxo-L-norleucine (DON) for 48 h either lone or in presence of AICAR. () Long term survivl of H1299 cells trnsfected with either control sirna or AMPKα1/α2 sirna nd cultured in DMEM contining either glucose or glutmine. Cells were grown for 48 h nd medium ws replced with fresh medium contining 25 mm glucose nd 2 mm glutmine nd further grown for 1 weeks. (c nd d) H1299 cells were grown in glucose-free DMEM contining 5 mm glutmine. Cells were treted with 5 M epiglloctechin 3-gllte (ECEG), 1 mm 6-dizo-5-oxo-L-norleucine (DON) or 1 M oligomycin in presence or sence of AICAR for 48 h. medium ws replced with drug free medium nd further grown for 1 week. Representtive imge of pltes stined with crystl violet (c) nd quntittive representtion of reltive intensity (d). 3
Supplementry figure S3 5mM Glucose 5mM Glutmine 5mM Glucose Control 35 No Sustrte Control 3 AICAR CC+AICAR 25 2 15 1 5 AICAR CC+AICAR c 35 No Sustrte Control 3 AICAR CC+AICAR 25 2 15 1 5 5mM Glutmine Supplementry figure S3: AMPK regultes glucose metolism under metolic stress (c) H1299 cells were cultured in DMEM contining either 5 mm glucose or 5 mm glutmine with or without AICAR nd compound C for 48 h. () Representtive imge of cells, ( nd c) represents percent cell survivl fter the tretment of indicted regents for 48 h. (Scle r = 5 μm). All vlues re represented s men ± SD. 4
Supplementry figure S4 WT-MEF Control Oligomycin Control Oligomycin Complete Glutmine AMPKα1/α2 -/- MEF Control Oligomycin Control Oligomycin Complete Glutmine No Sustrte Lctte No Sustrte Lctte Glucose Glctose Glucose Glctose Supplementry figure S4: AMPK regulted mitochondril metolism of non-glucose cron source like lctte nd glutmine. Representtive imge showing the morphology nd cell survivl of WT nd AMPK-DKO cells cultured in presence or sence of 5 mm glucose, 5 mm glutmine, 5 mm glctose nd 1 mm lctte with or without 1 M oligomycin for 48 h. (Scle r = 5 μm) 5
Supplementry figure S5 MFI (AU) 25 WT DKO 2 15 1 5 MEF MFI (AU) 15 EV DN 125 1 75 5 25 H1299 AIC - + - + - + - + Rp - - + + - - + + 25 mm Glu 1 mm Glu AIC - + - + - + - + Rp - - + + - - + + 25 mm Glu 1 mm Glu Supplementry figure S5: AMPK increses mitochondril density in cncer cells y suppressing mtor. () MEF (WT nd AMPK-DKO) nd () H1299 (EV nd DN) cells were cultured in either 25 mm or 1 mm glucose, nd 2 mm glutmine in presence or sence of.5 mm AICAR or 2 nm rpmycin either lone or together for 24 h. Mitochondril density ws mesured using Mitotrcker Red FM y flow cytometry. All vlues re represented s men ± SD. 6
Supplementry figure S6 % Vile cells 15 125 1 75 5 25 Veh ctrl AIC SB1 SB1 + AIC SB2 SB2 + AIC ATP level (% of control) 25 Veh ctrl AIC SB23 SB23 + AIC 2 SB2 SB2 + AIC 15 1 5 c + AICAR -AICAR SB2358 - + - SB2225 - - + d Reltive intensity (% of control) 3 Ctrl AIC SB2358 25 SB2358 + AIC SB2225 SB2225 + AIC 2 15 1 5 Supplementry figure S6: Inhiition of p38mapk prevents AMPK induced cell survivl under metolic stress. () H1299 cells were cultured in DMEM contining 1 mm glucose nd 2 mm glutmine in presence or sence of 25 M SB2358 nd SB2225 either lone or with.5 mm AICAR for 48 h. Cell viility ws ccessed y PI stining vi flow cytometry. () ATP level in H1299 cells grown under the condition mentioned in (). (c nd d) Long term cell survivl in H1299 cells grown in presence of 25 M SB2358 nd SB2225 either lone or with.5 mm AICAR for 24 h. Medium ws replced with drug free medium nd further grown for 1 week. Representtive imge showing long term survivl of H1299 cells (c), quntittive representtion of intensity of stined cells (d). All vlues re represented s men ± SD. 7
Supplementry figure S7 H1299 38 kd IB: p-p38 38 kd IB: p38 9 kd IB: PGC1-25 kd IB:TFAM 17 kda IB:COXIV 6 kda IB:HSP6 Rpmycin - + - + SB2358 - - + + Supplementry figure S7: Rpmycin increses levels of molecules involved in mitochondril iogenesis. Immunolots showing the levels of indicted molecules in H1299 cells cultured in glucose limiting condition with or without 2 nm rpmycin nd 1 M p38 specific inhiitor SB2358 for 24 h. 8
Supplementry Tle S1 Gene Sequence h Actin hpgc1α hcox5 F 5'-CATGTACGTTGCTATCCAGGC-3' R 5'-CTCCTTAATGTCACGCACGAT-3' F 5'-CTGCTAGCAAGTTTGCCTCA-3' R 5'-AGTGGTGCAGTGACCAATCA-3' F 5'-ATGGCTTCAAGGTTACTTCGC-3' R 5'-CCCTTTGGGGCCAGTACATT-3' 9