Figure 4. MPTP opening is involved in Bax mediated apoptosis.

(A) Heat stress induces MPTP opening as evidenced by the gradual decrease in cytosolic calcein. Heat stress treated HUVEC cells were stained with the calcein-AM and COCl₂ at equal volume mixture, and analyzed for fluorescence intensity using LSCM. (B) MPTP opening induced by heat stress is not affected by PFT inhibition of p53. Cells were pretreated with 20 μM BAPTA-AM and PFT, respectively, and then incubated at 43 °C for 2 h, and then further incubated for 1 h. The MPTP opening was measured by staining with calcein-AM and COCl₂. (C) Calpain activitiy is not affected in heat stress induced HUVEC cells. Calpain activities were measured by analyzing the fluorometric cleavage of their substrates Ac-LLY-AFC. HUVEC cells treated with 250 μM Dibucainewere as a positive control. (D,E) Cells were pretreated with 50 μM CsA, then incubated at 43 °C for 2 h, followed by further incubation for 6 h, and (D) Inhibition of MPTP opening diminishes the percentage of early apoptosis in heat treated HUVEC cells. Apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI. (E) Inhibition of MPTP opening reduces heat stressed induced Bax conformational change and mitochondrial translocation. Bax mitochondrial translocation was measured by western blot. Bax conformational change was assessed by immunoprecipitation. Data are presented as means ± SD of three separate experiments. *p < 0.05 as compared to control; ^#p < 0.05 as compared to heat stress (6 h). ^##p < 0.05 as compared to heat stress (1 h).