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. 2023 Mar 11;14(3):193. doi: 10.1038/s41419-023-05719-x

Fig. 4. Downregulation of PDIA4 suppresses ATF4/SLC7A11/GPX4 in RCCs.

Fig. 4

RNA-seq was performed in stable PDIA4-silencing-down 786-O cells, with scramble control 786-O cells as control. A Scatter plot of differential expression genes. B Overlay of downregulated genes (shPDIA4 vs scramble control) with known regulatory genes in ferroptosis. C Real-time PCR and western blot assay for SLC7A11 expression in stable PDIA4-silencing-down or scramble control 786-O cells. Stable PDIA4-silencing-down or scramble control 786-O cells were treated with Sal (2 µM). D Western blot assay of cell lysates using antibodies against p-PERK/PERK and ATF4. Stable PDIA4- or control MCS-overexpressing 786-O cells were treated with Sal (2 µM) for 24 h. E, F Western blot assay of cell lysates using antibodies against SLC7A11, PDIA4, phosphorylated PERK, and ATF4. 786-O cells were transfected with ATF4 overexpressing plasmid or control plasmid in the presence with or without Sal (2 µM). G Cell viability assay and H level of lipid peroxidation measured by MDA assay. I Western blot assay of PDIA4, PERK, ATF4, and SLC7A11 in cancerous and adjacent normal tissue resected from patients of RCC, and the statistical analyses. Data are present as mean ± S.E.M., n = 5. Student’s t-test was performed in (C) and (I), and one-way ANOVA analysis was performed in (G) and (H). *P < 0.05; **P < 0.01; ***P < 0.001.