Fig 1: Compound 6d induces mitochondrial-mediated intrinsic apoptosis by targeting Bcl-2. (A) CRC cells were treated with indicated concentrations of compound 6d for 8 h and then stained with the Annexin V-FITC/PI kit. The stained cells were analyzed with flow cytometry analysis. (B) Compound 6d regulates the expression of apoptosis-related proteins. CRC cells were treated with different concentrations of compound 6d (0, 5, 10, 15 µmol/L) for 8 h. The release of cytosol cytochrome c, expression of the BCL2 family proteins Bcl-2 and Bax, the cleavage levels of activated caspase 3 and poly (ADP-ribose) polymerase (PARP), were estimated to determine whether or not the intrinsic apoptotic pathway is involved in the anti-tumor effect. ß-actin level was used as internal control for equal protein loading. (C) Compound 6d treatment increased cytosolic cytochrome c levels. Control and compound 6d (15 µM)-treated cells were subjected to cell fractionation using kit to separate cytoplasmic and mitochondrial fraction, and then cytosolic and mitochondrial cytochrome c levels were detected through western blotting, respectively. The translocase of outer mitochondrial membrane 20 homolog (Tomm 20) was used as a mitochondrial marker. (D) Compound 6d inhibits the interaction between exogenous Bcl-2 and endogenous Bax, but not the interaction of Bcl-xL and Bax. After treatment, lysates from HCT116 cells transfected with HA-Bcl-2 or HA-Bcl-xL were pulled down with anti-HA, followed by WB with corresponding primary antibodies. **** p < 0.0001 versus vehicle. (E) In vitro binding assay was performed using recombinant His-Bcl-2 and His-Bax proteins. Anti-Bcl-2 antibody was used for pull-down after incubating recombinant proteins with compound 6d, AT101 and GX15-070, and then anti-His antibody was employed for WB. Both AT101 and GX15-070 were used as positive control.