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
EphB4/CDC42/N-WASP signaling plays a role in PRIS-induced cell death in CRLCs. (a) Protein expression of EphB4 was detected by Western blot in BEAS-2B cells and CRLCs derived from the tumor tissue of different patients. (b) CRLCs were treated with 0 (control), 2, 4, and 8 μM PRIS for 24 h; cell lysates were resolved by SDS-PAGE and analyzed by Western blot with antibodies against EphB4, Ephrin-B2, CDC42, N-WASP, and GAPDH. (c) Cells were treated as described in (b); EphB4 and N-WASP were stained with primary antibodies followed by staining with secondary DyLight® 594-conjugated antibodies. Cell nuclei were labeled with DAPI, and immunofluorescence analysis was performed under the fluorescence microscope. (d, e) PRIS (4 μM) alone or combined with NVP-BHG712 (0.1 μM) was applied to CRLCs, and Western blot analysis and cellular viability assay were performed. (f) CRLCs were transfected with EphB4-specific and N-WASP-specific or nonspecific siRNA for 48 h. mRNA and protein expression was measured to determine the efficiency of the silence. (g) CRLCs were incubated in the absence or presence of EphB4 siRNA for 48 h. Then, cells were treated by PRIS (4 μM) and indicated protein expressions were measured by Western blot. (h, k) Cells were transfected with scrambled (Scr) control, EphB4, or N-WASP siRNA for 48 h and then treated with 4 μM PRIS for 24 h; cell viability was determined by MTS assay (). (i, l) CRLCs were treated under the same conditions as described above in (h, k), and cell migration and invasion ability were determined by Transwell assay. The numbers of migrated cells were quantified by performing cell counts of 10 random fields (). (j, m) CRLCs were treated under the same conditions as described above in (h, k); HUVECs treated by conditioned medium were seeded on a Matrigel-precoated 96-well plate, and capillary-like tube formation was observed under an inverted light microscope (). All Western blot band intensities were normalized to GAPDH. Data are presented as ,,. ns: not significant.
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