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. 2021 Jul 11;46:102065. doi: 10.1016/j.redox.2021.102065

Fig. 3.

Fig. 3

IDH1 and ME1 play a key role in maintaining redox balance and resisting cell death in 4T1 cells under glucose-deprivation with or without lactic acidosis

(A) Cellular GSH/GSSG ratios of 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. (B) Cellular ROS levels of 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. ROS was probed by DCFH-DA. Bar = 25 μm. (C) The redox state of thioredoxin 1 (Trx1) in 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. The reduced form of Trx1 was derivatized with IAA, hence it had more negative charge than oxidized form and moved faster in the native PAGE. The reduced form/oxidized form ratios were calculated based on the gray values of the bands. (D) The redox state of peroxiredoxin 1 (Prx1) in 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. The reduced form of Prx1 is monomer and the oxidized form is dimer, hence they can be separated by non-reducing SDS-PAGE. The reduced form/oxidized form ratios were calculated based on the gray values of the bands.(E) Survival curves of 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. (F) The necrosis and apoptosis rates of 4T1 control, IDH1 knockout, ME1 knockout and IDH1&ME1 knockout cells under glucose deprivation with or without lactic acidosis. PI (+), Annexin V-FITC(+): necrosis or late apoptosis; PI (−), Annexin V-FITC(+): early apoptosis. Glc (−): Glucose-free RPMI-1640 medium; Glc (+) + LA: Glucose-free RPMI-1640 medium supplemented with 20 mM lactic acid, pH 6.7. Data are shown as means ± SD, with n = 3 biological replicates in (A) (C) (D) (F), n = 5 biological replicates in (E), and the results were all confirmed by three independent experiments. n. s. not significant, **p < 0.01, ***p < 0.001 (Student's test). See also Supplementary Figs. 4 and 5.