DPV022 inhibits Bax-induced apoptosis and Bax oligomerization. (A) HeLa cells were cotransfected with pEGFP, pEGFP-DPV022, pEGFP-FPV039, pEGFP-M11L, or pEGFP-Bcl-2 and pcDNA3-HA-Bax. Apoptosis was assessed by quantifying TMRE fluorescence via flow cytometry, and the percentage of cells that demonstrated a loss of mitochondrial membrane potential (ΔΨm) is given on the y axis. Standard deviations were calculated from three independent experiments. (B) HeLa cells were transfected, and the expression level of each EGFP-tagged construct was assessed by Western blotting (WB). (C) HEK 293T cells were cotransfected with pEGFP, pEGFP-Bcl-2, or pEGFP-DPV022 and pcDNA3-HA-Bax. zVAD.fmk was added after transfection to prevent the activation of caspases. Following lysis in 2% CHAPS buffer, cells were treated with 1 μM BMH and Western blotted (WB) with anti-HA to detect Bax oligomers (top). A portion of the lysates (input) was treated with dimethyl sulfoxide (DMSO) alone and subjected to Western blotting with either anti-HA or anti-EGFP antibodies to determine expression levels of the transfected proteins (middle and bottom). ×1, Bax monomer; ×2, Bax dimer; ×N, higher-order Bax oligomers.