Fig. 6

Drp1 is required for CPT-mediated apoptosis. a Immuno-staining of Bax and Drp1. Confocal microscope scanning photographs indicated the mitochondrial localization of Drp1 and Bax in CPT treated 143B cells. Red: Mito-Tracker Red CMXRos, Green: Bax and Drp1 as indicated. b Expression of Drp1 and Bax in cytosolic and mitochondrial fractions in OS cells were determined by immunoblotting using anti-Drp1 and anti-Bax antibodies. c Interaction between Bax and Drp1 after CPT treatment. Proteins were extracted from CPT-treated OS cells, and then IP was performed with Bax antibody; Co-IP Drp1 and Bax was detected by Western blotting. d After knocking down Drp1 for 24 h, OS cells were treated with CPT 20 μM for indicated time. Bax expression was determined in both cytosolic and mitochondrial fractions using mouse anti-Bax antibody. Hsp60 and α-tubulin antibodies were used as loading controls for mitochondria and cytosol, respectively. e 143B cells were pretreated with Bax inhibitor peptide V5 (BIP-V5) (50 or 200 μM) for 1 h and incubated with 20 μM CPT for 24 h. Annexin V/PI analysis was used to detect apoptotic cells by flow cytometry followed by CPT treatment. The respective cell percentages in early and late apoptosis for different dose treatment are presented in the qantitative analysis. The results were expressed as the means ± SD from three independent experiments. *P < 0.05, significantly different compared with control