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. 2018 Dec 27;104(1):179–185. doi: 10.1016/j.ajhg.2018.11.018

Figure 3.

Figure 3

Increased ER Stress-Induced Apoptosis in RNF13-Mutant Cells

(A) Control fibroblasts and fibroblasts derived from affected individual 1 were analyzed for RNF13 protein abundance by immunoblot. To confirm that the indicated band corresponds to RNF13, we treated HeLa cells with RNF13-targeting siRNA and analyzed cells in parallel.

(B and C) Increased signaling of ER stress in fibroblasts (B) and lymphoblasts (C) derived from affected individual 1. Cells were treated with 1 μg/mL tunicamycin for 48 hr, and lysates were analyzed for XBP1 splicing (2.4- and 2.5-fold increases in the affected individual’s fibroblasts and lymphoblasts, respectively, when results were normalized against those for vinculin) and Jun phosphorylation at Ser73 (3.2- and 3.7-fold increases in the affected individual’s fibroblasts and lymphoblasts, respectively). The asterisk indicates a crossreactive band.

(D) Increased tunicamycin-induced XBP1 splicing in the affected individual’s lymphoblasts, quantified by qRT-PCR. Cells were treated with 1 μg/mL tunicamycin for 48 hr. The average of six experiments, with standard deviations indicated as error bars, is shown. Tunicamycin-treated samples were normalized to the “no drug” condition for each cell type. The statistical difference between tunicamycin-treated control cells and those from the affected individual (TTEST, two-tailed, unequal variance) is indicated. Baseline (no drug treatment) amounts of spliced XBP1 mRNA are not significantly different in the affected individual (Figure S3A).

(E) Increased tunicamycin-induced apoptosis in the affected individual’s lymphoblasts. Cells were treated with 1 μg/mL tunicamycin for 48 hr and analyzed for Annexin V labeling by flow cytometry. The average of four experiments, for which standard deviations are indicated as error bars, is shown. Tunicamycin-treated samples were normalized to the “no drug” condition for each cell type. The statistical difference between tunicamycin-treated control cells and the affected individual’s cells (TTEST, two-tailed, unequal variance) is indicated. Baseline (no drug treatment) levels of apoptosis are not significantly different in the affected individual (Figure S3B).