Validation of Prdx1 as a target of CM728 in TNBC cells. A, Identification of Prdx1 as a target of CM728 in MDA-MB-231 cells by western blotting. Cell extracts were used as a positive control. B, Cell extracts of Prdx1 knockdown MDA-MB-231 and BT-549 cells were used to evaluate the expression levels of Prdx1 by immunoblotting. Calnexin was used as a loading control (shC: shRNA transfection control; sh#10, sh#11, and sh#12: shRNA sequences targeting Prdx1). C, Effect of Prdx1 downregulation on the percentage of DCF fluorescence in MDA-MB-231 cells untreated or treated with 1 µM CM728 for 30 min. D, Effect of Prdx1 knockdown on MTT metabolism of MDA-MB-231 and BT-549 cells, untreated or treated with 0.2 or 0.15 µM CM728, respectively, at 48 h. E, Western Blot analysis of the monomer (M) and dimer (D) forms of Prdx1, or the levels of SO2/3-Prdx in response to CM728 (1 µM) in MDA-MB-231 cells. β-actin was used as a loading control. Densitometry quantification from immunosignal values is shown below the bands. Values are relativized to the loading control. Representative images of two independent experiments are shown. Statistical analyses were conducted using one-way ANOVA followed by Dunnett's multiple comparison test. *, P < 0.05 versus vehicle-treated shC cells; ***, P < 0.0001 versus vehicle-treated shC cells; ##, P < 0.001 versus CM728-treated shC cells; ###, P < 0.0001 versus CM728-treated shC cells.