Fig. 2.

GPD2 suppresses mitochondrial lipid peroxidation. (A) Western blot showing GPD2 protein levels in cytosolic and mitochondrial fractions from GPD2-KO cells expressing the indicated GPD2 constructs. mito, mitochondrial; cyto, cytosolic. (B–E) Mitochondrial lipid peroxidation measurement by flow cytometry or immunofluorescence in Cas9 control and GPD2-KO cells upon treatment with RSL3 (1 μM) for 2 h (B–D) or 24 h (E). (F–H) Results of PI staining of GPD2-KO HeLa (F), RPMI 7951 (G), and HCT116 (H) cells expressing the indicated GPD2 constructs upon treatment with RSL3 (10 μM). (I–L) Mitochondrial lipid peroxidation measurement by flow cytometry or immunofluorescence in GPD2-KO HeLa (I and J), RPMI 7951 (K), and HCT116 (L) cells expressing the indicated GPD2 constructs upon treatment with RSL3 (1 μM). (M) Cell viability measurement of HeLa cas9 control cells with RSL3, TEMPO, or MitoTEMPO treatment as indicated. (N–P) Results of PI staining of Cas9 control HeLa (N), RPMI 7951 (O), and HCT116 (P) cells treated with RSL3 (10 μM) for 6 h (N and O) or 24 h (P) following pretreatment with a vehicle, TEMPO (10 μM), MitoTEMPO (10 μM), or ferrostatin-1 (Ferr-1; 5 μM) for 24 h. (Q–S) Mitochondrial lipid peroxidation measurement in GPD2-KO HeLa (Q), RPMI 7951 (R), and HCT116 (S) cells treated with RSL3 (1 μM) for 2 h (Q and R) or 24 h (S) following pretreatment with a vehicle, TEMPO (10 μM), MitoTEMPO (10 μM), or Ferr-1 (5 μM) for 24 h. (T) Cell viability measurement of HeLa GPD2-KO cell with RSL3, TEMPO, or MitoTEMPO treatment as indicated. (U–W) Results of PI staining of GPD2-KO HeLa (U), RPMI 7951 (V), and HCT116 (W) cells treated with RSL3 (10 μM) for 6 h (U and V) or 24 h (W) following pretreatment with a vehicle, TEMPO (10 μM), MitoTEMPO (10 μM), or Ferr-1 (5 μM) for 24 h. Data are presented as means (± SD) for three independent repeats. Unpaired, two-tailed t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.