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. 2014 Mar 26;4:4469. doi: 10.1038/srep04469

Figure 5. Heat stress-induced apoptosis in P53 siRNA transfectant HUVEC cells.

Figure 5

(a) HUVEC cells were transfected with scrambled siRNA (Scr) or P53 siRNA (P53). Western Blots of P53 protein expressed(cropped) in HUVEC transfectant cells.β-actin was run as an internal control. (b) RT-PCR analysis of P53 in HUVEC transfectant cells. Total RNA was isolated from HUVEC transfectant cells and P53 mRNA levels were determined by RT-PCR and analyzed on agarose gel electrophoresis. β-actin from the same samples was amplified as control. (c–f) HUVEC transfectant cells were treated with 43°C for 2 h with the cells further incubated for 6 h, and (c) apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining. (d) Enzymatic activity of caspase-9 was measured in cell lysates using fluorogenic substrate Ac-LEHD-AFC and was expressed relative to the control at 37°C (100%).(e) The loss of ΔΨm was measured by JC-1 and flow cytometry. (f) Intracellular location of cytochrome C was determined by Western Blots(cropped). COX IV, mitochondrial loading control. Each value is the mean ± SD of three separate determinations. *P < 0.05 versus scrambled siRNA-transfected cells exposed to heat stress.