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

Fig. 3.

Fig. 3.

The IRE1α/XBP1 and PERK/eIF2α/ATF4 axes of the ER stress response pathway are activated in OC cells under restricted availability of UFA. (A) XBP1 splicing (u, unspliced transcript; s, spliced transcript) 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) and cultured in low-serum conditions (1% FBS) for 48 h. (B) Densitometric analysis of XBP1 splicing products shown in A. Bars represent percentage of spliced XBP1 relative to total XBP1 (mean ± SD, n = 3). (C) XBP1 splicing in OVCAR-5 cells cultured in low-serum conditions and treated with SCD inhibitor CAY10566 for 48 h. (D) Percentage of spliced XBP1 isoform measured by densitometric analysis of PCR products shown in C (mean ± SD, n = 3). (E) XBP1 splicing and (F) Western blot of proteins of the PERK/eIF2α/ATF4 axis in shCtrl and shSCD OVCAR-5 cells cultured under low-serum conditions and treated with 50 μM PA for the time periods indicated. Arrows indicate the band of interest. (G) XBP1 splicing in primary cells from tumors of OC patients cultured in low-serum conditions and treated with 3 μM CAY10566 for 48 h or with 1 μM CAY10566 and 50 μM PA for 12 h. (H) Representative SRS images in the C-H and C-D regions of OVCAR-5 shCtrl and shSCD cells cultured in low-serum conditions (1% FBS) and treated with 12.5 μM PA-d31, with or without 52 μM OA, or cultured in medium with full serum (10% FBS) and treated with PA-d31 for 24 h. Yellow arrows indicate rigid ER, gray arrows indicate LDs, and blue arrows indicate cytoplasm. (Scale bar, 20 µm.) (I) Percentages of shCtrl and shSCD OVCAR-5 cells treated as described in H showing C-D SRS signal mainly in rigid ER, LD, and cytoplasm (n = 139 to 191). (J) TEM imaging of smooth ER (donut shaped, red arrows; ski board shaped, blue arrows) in OVCAR-5 shCtrl vs. shSCD cells cultured in low-serum conditions for 48 h. Other organelles are highlighted (M, mitochondria; N, nucleus; NL, nucleolus). **P < 0.01, ***P < 0.001, ****P < 0.0001.