Fig 2: Effects of the CSE knockdown induced by transfection with si-CSE on the trimetazidine-induced protection of H9c2 cells against H/R-induced injury. H9c2 cells were transfected with si-CSE or si-scramble and exposed to trimetazidine (10 µM) for 1 h prior to H/R treatment. (a) Determination of CSE protein expression by western blot analysis, reflecting the transfection efficiency of si-CSE. (b) Quantitative analysis of the western blotting result. (c) Measurement of the viability of H9c2 cells by the Cell Counting Kit-8 assay. (d) Detection of LDH activity in the cell supernatant by the LDH cytotoxicity detection kit. The values represent the mean±standard error of the mean (n=3). **P<0.01 vs. control group; ##P<0.01 vs. H/R treatment group; $P<0.05 and $$P<0.01 vs. trimetazidine and H/R co-treatment groupCSE - cystathionine ?-lyase; si-CSE - specific siRNA against human CSE; si-scram/si-scramble - scrambled siRNA; H/R - hypoxia/reoxygenation; LDH - lactate dehydrogenase

Fig 3: Effects of CSE knockdown on oxidative stress in the presence or absence of trimetazidine in H/R-treated H9c2 cells. H9c2 cells were transfected with si-CSE or si-scramble and exposed to trimetazidine (10 µM) for 1 h prior to H/R treatment. (a) Determination of intracellular ROS production using probe 2’,7’-dichlorofluorescin diacetate. The green fluorescence in the cells was observed using a fluorescent microscope (x200). (b) Quantitative analysis of ROS production was carried out using an FACS Calibur flow cytometer. (c) Measurement of MDA content in H9c2 cells using a lipid peroxidation assay kit. (d) The expression of Nox2 protein by western blot analysis. (E) Quantitative analysis of Nox2 protein expression. (f) Detection of SOD activity using an SOD assay kit. One unit of SOD is defined as the amount of enzyme in protein sample solution (20 µl) that inhibits the reduction of WST-1 by superoxide anions by 50%. (g) Detection of GSH-Px level using a GSH-Px assay kit (colorimetric method). The values represent the mean±standard error of the mean (n=3). *P<0.05 and **P<0.01 vs. control group; #P<0.05 and ##P<0.01 vs. H/R treatment group; $P<0.05 and $$P<0.01 vs. trimetazidine and H/R co-treatment group.CSE - cystathionine ?-lyase; H/R - hypoxia/reoxygenation; ROS - reactive oxygen species; MDA - lipid peroxidation marker; Nox2 - NADPH oxidase 2; SOD - superoxide dismutase; GSH-Px - glutathione peroxidase

Fig 4: Effects of the CSE knockdown on the trimetazidine-induced inhibition of apoptosis in H/R-treated H9c2 cells. H9c2 cells were transfected with si-CSE or si-scramble and exposed to trimetazidine (10 µM) for 1 h prior to H/R exposure. (a) Analysis of the caspase-3 activity by a colorimetric assay kit. (b) Western blot analysis and quantification of the expression of apoptosis regulators (c) BAX and (d) Bcl-2 in H9c2 cells. The values represent the mean±standard error of the mean (n=3). **P<0.01 vs. control group; ##P<0.01 vs. H/R treatment group; $P<0.05 and $$P<0.01 vs. trimetazidine and H/R co-treatment group

Fig 5: Cysteine and cystine deprivation halt growth and reduce viability in glioma through reduced protein translation and glutathione synthesis. (A) Growth rate of IDH1-mutant glioma cell lines under cysteine/cystine (Cys/Cyss) deprivation conditions over 96 h. Data are shown as the ratio of viable cells to cells seeded (2-way ANOVA followed by Sidak’s multiple comparison test, *P < 0.05; **P < 0.005; ***P < 0.001; n = 4, data displayed as mean ± SD for each time point and condition). (B) Neurosphere formation after 96 h of incubation in the same medium. BT142 cells displayed as a representative picture (experiment was done in triplicate for each cell line); scale bar is 100 µm. (C) Viability after 96 h in medium lacking cysteine/cystine as the percentage over the viability of cells in control conditions (n = 3, data displayed as mean ± SD for each time point and condition). (D) Metabolic pathways related to cysteine (THF, tetrahydrofolate; MS, methionine synthase; CBS, cystathionine beta-synthase; CTH, cystathionine gamma-lyase; GCLM, glutamate–cysteine ligase modulatory subunits; GCLC, glutamate–cysteine ligase catalytic subunits). (E) Rescue experiments involving the addition of different metabolites and agents to cells grown without cysteine/cystine. Data displayed as control-normalized growth (top panel) and viability (bottom panel) as the mean ± SD for n = 3; *P < 0.05; **P < 0.005; ***P < 0.001; each condition was compared with the -Cys/Cyss values by using a two-tailed Student’s t-test. (F) Glutathione (GSH) levels in complete medium (+) and medium lacking cysteine/cystine (-) plus 0.1 mm homocysteine and cystathionine (2-way ANOVA followed by Dunnett’s multiple comparison test for -cysteine/cystine vs all groups, **P < 0.005; ***P < 0.001 was used and data are displayed as the mean ± SD for n = 3). (G) Representative western blot of p-GCN2 and p-eIF2a in both complete and cysteine/cystine-free media for the three cell lines investigated (quantitative data for n = 3 is displayed in Fig. S2C,D). (H) Intracellular ROS levels assessed by the DCFDA assay in glioma cell lines under cysteine/cystine deprivation and treated with 0.25 mm trolox for 96 h. BT142 cell line data are shown as a representative diagram and data from other cell lines are shown in the Fig. S1E.