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. 2022 Oct 5;119(41):e2203480119. doi: 10.1073/pnas.2203480119

Fig. 4.

Fig. 4.

UFA-induced ER stress response is reversed by exogenous OA. XBP1 splicing measured by RT-PCR and agarose gel electrophoresis in OVCAR-5 cells transduced with control shRNA (shCtrl) or shRNAs (1 or 2) targeting SCD (shSCD), cultured in medium containing low serum, and treated (A) with indicated doses of OA for 48 h or (B) with 50 μM PA and indicated doses of OA for 12 h. Western blot of SCD and proteins of the PERK/eIF2α/ATF4 axis in shCtrl and shSCD cells cultured in low serum medium and treated (C) with different doses of OA for 48 h or (D) with 50μM PA and indicated doses of OA for 12 h. XBP1 splicing in OVCAR-5 cells cultured in medium containing low serum and treated (E) with 21.6 nM CAY10566 and indicated doses of OA for 48 h or (F) with 8.1 nM CAY10566, 50 μM PA, and indicated doses of OA for 12 h. (G) Proteins of the PERK/eIF2α/ATF4 pathway measured by Western blot in OVCAR-5 cells treated as described in C. (H) Western blot of proteins of the PERK/eIF2α/ATF4 pathway in OVCAR-5 cells treated as described in D. Arrows indicate the band of interest. Verification of SCD overexpression by (I) qRT-PCR and (J) Western blotting in OVCAR-5 cells transduced with a SCD expression vector (pLenti-SCD). Cells transduced with empty vector (pLenti-Ctrl) served as control. Bars represent means ± SD, n = 3. (K) XBP1 splicing in pLenti-SCD vs. pLenti-Ctrl cells cultured in low-serum conditions and treated with 50 μM PA for 12 h. (L) Percentage of spliced isoform (mean ± SD, n = 3) calculated by densitometric analysis of PCR products shown in K. *P < 0.05, ***P < 0.001.