Oxidative Medicine and Cellular Longevity / 2020 / Article / Fig 5

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 5

PRIS induces ER stress in CRLCs. (a, b) CRLCs were treated with 0 (control), 2, 4, and 8 μM PRIS or 2 μM Tunicamycin (TM) for 24 h; cell lysates were resolved by SDS-PAGE and analyzed by Western blot with antibodies against CHOP, GRP78, ATF4, and GAPDH. (b) CRLCs were treated with 0 (control), 2, 4, and 8 μM PRIS or 2 μM TM for 6 h; cell lysates were resolved by SDS-PAGE and analyzed by Western blot with antibodies against p-eIF2α, eIF2α, p-IRE1α, and IRE1α. (c–e) Cells were pretreated with 4-PBA (1 mM) for 90 min and followed by treatment with 4 μM PRIS or 2 μM TM for 24 h. After each treatment, cells were incubated with 20 nM DiOC6 for 30 min and MMP was measured by flow cytometry (). Cellular viability was determined by MTS assay (). Cell lysates were analyzed by Western blot with antibodies against CHOP, GRP78, ATF4, cleaved caspase-4, and GAPDH. (f) CRLCs were transfected with CHOP-specific or nonspecific siRNA. 48 h after transfection, mRNA and protein expression was measured to determine the efficiency of the silence. (g) CRLCs were incubated in the absence or presence of CHOP siRNA for 48 h, then treated with PRIS (4 μM) or TM (2 μM), and cellular viability was determined by MTS assay (). (h–j) Cells were treated as described above in (g); production of ROS was quantified by the amount of cellular DCF synthesis. The fluorescence intensity was quantified using a fluorescence microplate reader (). MMP level was measured by flow cytometry (), and indicated protein expressions were detected by Western blot. (k) CRLCs were transfected with CHOP-specific or nonspecific siRNA, then incubated with PRIS (8 μM) for 24 h. Vehicle- or PRIS-treated cells were stained with Annexin V-FITC and PI and evaluated by flow cytometry (). All Western blot band intensities were normalized to the total proteins or GAPDH. Data are presented as , , .
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)

Article of the Year Award: Outstanding research contributions of 2020, as selected by our Chief Editors. Read the winning articles.