Oxidative Medicine and Cellular Longevity / 2020 / Article / Fig 8

Research Article

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 8

PRIS induces ER stress-mediated intrinsic cell apoptosis via EphB4/CDC42/N-WASP signaling. (a) CRLCs were incubated in the absence or presence of EphB4 siRNA for 48 h. Then, cells were treated by 4 μM PRIS and indicated protein expressions were measured by Western blot. (b, c) CRLCs were treated under the same conditions as described above in (a); ROS generation was determined by the amount of cellular DCF formation (), and MMP level was detected by flow cytometry (). (d, e) Cells were transfected with scrambled (Scr) siRNA or EphB4 siRNAs for 48 h and then treated with 4 μM PRIS for 24 h. Relative changes in cytochrome c, cleaved caspase-9 and caspase-3, Bcl-2, and Bax protein levels were analyzed by Western blot. (f) CRLCs were incubated in the absence or presence of N-WASP siRNA for 48 h. Then, cells were treated by 4 μM PRIS and indicated protein expressions were measured by Western blot. (g) Cells were treated as described above in (f); production of ROS was quantified by the amount of cellular DCF synthesis. The fluorescence intensity was quantified using a fluorescence microplate reader (). (h, i) Cells were transfected with scrambled (Scr) siRNA or N-WASP siRNA for 48 h and then treated with 4 μM PRIS for 24 h. Relative changes in Bcl-2, Bax, and cleaved caspase-3 protein levels were analyzed by Western blot. All Western blot band intensities were normalized to GAPDH or α-tubulin. Data are presented as , , . ns: not significant.
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