Effect of WA on the proliferation and oxidative stress of PHGDH KO HCT-116 cells. (A) Growth curves of PHGDH KO HCT-116 cells after treatment with or without WA (2 μmol/L). Data are presented as the mean ± SD, n = 6, ns, no significance versus PHGDH KO group. (B) Cell proliferative activity of PHGDH KO HCT-116 cells after treatment with or without WA (2 μmol/L) by the colony formation assay. Data are presented as the mean ± SD, n = 3, ns, no significance versus PHGDH KO group. (C) The ROS levels in PHGDH KO HCT-116 cells after treatment with WA (2 μmol/L) were measured by flow cytometry. Data are presented as the mean ± SD, n = 3, ∗P < 0.05 versus PHGDH KO group. (D) The ROS levels in PHGDH KO HCT-116 cells after treatment with WA (2 μmol/L) were measured by fluorescence microscope (DCFH-DA staining). Scale bar = 50 μm. (E) GSH/GSSG ratio of PHGDH KO HCT-116 cells after treatment with WA (2 μmol/L) were measured by a Glutathione Assay Kit. Data are presented as the mean ± SD, n = 3, ∗P < 0.05 versus PHGDH KO group. (F) NADPH/NADP+ ratio of PHGDH KO HCT-116 cells after treatment with WA (2 μmol/L) were performed by a NADPH assay kit. Data are presented as the mean ± SD, n = 3, ns, no significance versus PHGDH KO group. (G) The expression of γH2AX, CHK2, cyclin D1, cleaved caspase3, and cleaved PARP in PHGDH KO HCT-116 cells were measured by immunoblotting after treatment with WA (2 μmol/L).