Fig. 3.

SIRT1 promoted RSL3-induced downregulation of SLC7A11 and GPX4. (A) Western blotting showed that RSL3 (10 μmol/L) induced time-dependent downregulation of SLC7A11 and GPX4 in U87, U251 and U118 glioma cells. (B and C) RSL3-triggered declines of cysteine and GSH became more apparent in the cells pretreated 1 h with SRT2183 (20 μmol/L), but suppressed in the cells pretreated with EX527 (200 μmol/L). (D) Western blotting revealed SRT2183 exacerbated, but EX527 attenuated RSL3-induced downregulation of SLC7A11 and GPX4. (E)Western blotting demonstrated silence of SIRT1 with siRNA prevented the downregulation of SLC7A11 and GPX4 caused by RSL3 (10 μmol/L). (F and G) Knockdown of SIRT1 with siRNA partially reversed RSL3-indcued declines of cysteine and GSH. (H) Western blotting showed treatment with SRT2183 (40 μmol/L) could obviously upregulate SIRT1 in both cytoplasmic and nuclear fractions, but downregulate acetyl-p53, SLC7A11 and GPX4 in a time-dependent manner. (I) LDH release assay showed the glioma cell death caused by SRT2183 (40 μmol/L) at 24 h was attenuated when the cells were pretreated 1 h with EX527 (200 μmol/L) or supplemented with exogenous NAD+ (2 mmol/L). (J and K) The declines of cysteine and GSH induced by SRT2183 (40 μmol/L) at 24 h were both suppressed by pretreating the cells 1 h with EX527 or supplementing exogenous NAD+. (L) Western blotting proved pretreatment with EX527 or supplement of exogenous NAD + for 1 h not only alleviated SIRT1 upregulation in both cytoplasmic and nuclear fractions, but also inhibited the downregulation of acetyl-p53, SLC7A11 and GPX4 provoked by SRT2183 (40 μmol/L) at 24 h. (M) NAD + assay showed that NAD + decline triggered by SRT2183 (40 μmol/L) at 24 h was inhibited in the cells pretreated 1 h with EX527 or supplemented with exogenous NAD+. *: p < 0.01 versus control group; #: p < 0.01 versus RSL3 group; &: p < 0.01 versus SRT2183 group The values are expressed as mean ± SD (n = 5 per group).