Pristimerin Exacerbates Cellular Injury in Conditionally Reprogrammed Patient-Derived Lung Adenocarcinoma Cells by Aggravating Mitochondrial Impairment and Endoplasmic Reticulum Stress through EphB4/CDC42/N-WASP Signaling

Figure 4

PRIS induces caspase-dependent intrinsic apoptotic cell death in CRLCs. (a) CRLCs were treated with 0 (control), 2, 4, and 8 μM PRIS for 12 h; caspase assay was performed to measure the activity of caspase-9, caspase-3, and caspase-4 (). (b) Cells were treated as described above in (a); relative changes of protein levels in cytochrome c, cleaved caspase-9, cleaved caspase-3, and cleaved caspase-4 were analyzed by Western blot. (c) Cells were incubated with caspase inhibitors (10 μM Z-LEHD-FMK for caspase-9, 10 μM Z-DEVD-FMK for caspase-3, and 10 μM Ac-LEVD-CHO for caspase-4) for 1 h, followed by treatment with PRIS (4 μM) for 24 h. Cell viability was measured by MTS assay (). (d) CRLCs were transfected with caspase-4-specific or nonspecific siRNA for 48 h, and mRNA and protein expression was measured to determine the efficiency of the silence. (e, f) CRLCs were incubated in the absence or presence of caspase-4 siRNA for 48 h. Then, cells were treated by PRIS (4 μM) (or not) and cell viability or protein expression was determined by MTS assay () or Western blot. (g) CRLCs were incubated in the absence or presence of caspase-4 siRNA for 48 h. Then, cells were treated by PRIS (8 μM) and cell apoptosis was assessed by flow cytometry using Annexin V/PI double staining. (h, i) Cells were treated with scramble siRNA or siRNA to caspase-4 for 48 h and then exposed to PRIS (8 μM) for 24 h. Caspase activity was determined using specific substrates (). (j) CRLCs were preincubated with caspase inhibitors (10 μM Z-LEHD-FMK for caspase-9, 10 μM Z-DEVD-FMK for caspase-3, and 10 μM Ac-LEVD-CHO for caspase-4) for 1 h, followed by treatment with PRIS (8 μM) for 24 h. The activities of caspase-4 were monitored via caspase assay (). All Western blot band intensities were normalized to GAPDH. Data are presented as , , .

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