Bak knockout inhibited TNFα/CHX-induced PARP cleavage generation, caspase-3 activation, cytochrome C release, and Bax translocation. After induction of TNFα and CHX for 6 h, CRISPR control cells (#5) and Bak-knockout cells (#12, #16, and #15) were harvested and divided into two batches. One batch of cells were directly lysed and subjected to SDS–PAGE, in turn followed by Western blotting. The other batch was used for preparation of cytosolic and mitochondrial fractions using the Mitochondria Isolation Kit. (A) Bak expression (panel 1), PARP cleavage (panel 2), and caspase-3 activation (panel 3) in various cells is shown. Membranes in top panels were reprobed with β-actin antibody to indicate relative samples loading (bottom panels). (B) CRISPR control cells (#5, row 1) and Bak-KO cells (#12, #16, and #15, rows 2–4) were double stained with annexin V and PI. Fluorescence was detected using a flow cytometer to analyze necrotic (annexin−/PI+, quadrant 1), nonapoptotic (annexin−/PI−, quadrant 3), early apoptotic (annexin+/PI−, quadrant 4), and late apoptotic cells (annexin+/PI+, quadrant 2) in the presence (right column) or absence (left column) of TNFα and CHX for 6 h. (C) Cytochrome C (panel 1) from the mitochondria into the cytosol and Bax translocation (panel 2) from the cytosol into the mitochondria are shown. Membranes in top panels were reprobed with COX I antibody for confirming the mitochondrial integrity during experimental (panel 3). Membranes of second panel were reprobed with β-actin antibody to indicate relative sample loading of cytosolic fractions (bottom panels).