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. 2022 Jun 24;119(26):e2121987119. doi: 10.1073/pnas.2121987119

Fig. 1.

Fig. 1.

Metabolomic analysis links GPD2 to ferroptosis regulation. (A–C) Volcano plots comparing the metabolomic profiles of HT1080 cells treated with a vehicle, RSL3 (A; 10 μM), ML210 (B; 10 μM), or ML162 (C; 10 μM) for 2 h. (D) Fold changes in G3P levels in HT1080 cells upon treatment with RSL3 (10 μM), ML210 (10 μM), or ML162 (10 μM) for 2 h. (E) Cell viability measurement of HCT116 cells treated with RSL3 at different concentrations for 24 h following pretreatment with a vehicle or G3P (1 mM) for 24 h. (F) Schematic of G3P metabolism. (G and H) RSL3 resistance correlates with high GPD2 expression (G), and high GPD2 expression correlates with resistance to treatment with GPX4 inhibitors (RSL3, ML162, and ML210) in cancer cells (H). The plotted data were mined from the CTRP database. The plotted values are Pearson’s correlation coefficients. The scatter plots show the medians, 10th and 90th percentiles, and minima and maxima of the distributions. (I) Western blot of GPD2, ACSL4, FSP1, SLC7A11, DHODH, and GPX4 protein levels in Cas9 control and GPD2-KO cells. (J) Cell viability measurement of HeLa GPD2-KO and control cell after RSL3 treatment. (K–P) Results of PI staining of Cas9 control and GPD2-KO (numbers 1 and 2) HeLa (K and N), RPMI 7951 (L and O), and HCT116 (M and P) cells treated with RSL3 (10 μM), ML210 (10 μM), or ferrostatin-1 (Ferr-1; 5 μM) as indicated. HeLa and RPMI 7951 cells were treated for 6 h, and HCT116 cells were treated for 24 h. (Q–S) Lipid peroxidation measurement in Cas9 control, GPD2-KO HeLa (Q), and RPMI 7951 (R) cells treated with RSL3 (10 μM) for 3 h and HCT116 cells (S) treated with RSL3 (1.5 μM) for 24 h. (T and U) Cell viability measurement of HeLa control (T) and GPD2-KO (U) cells after G3P and RSL3 treatment. (V and W) Results of PI staining of Cas9 control and GPD2-KO HeLa (V) and RPMI 7951 (W) cells treated with RSL3 (10 μM) for 4 h following pretreatment with a vehicle or G3P (1 mM) for 24 h. Data are presented as means (± SD), n = 3 independent repeats. Unpaired, two-tailed t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.