Heightened ISR in <i>EIF2B4</i>^<i>A392D</i> cells.

<p>(A) Flow cytometry analysis of <i>CHOP</i>::<i>GFP</i> and <i>XBP1</i>::<i>Turquoise</i> dual reporter-containing parental CHO-S21 and <i>EIF2B4</i>^<i>A392D</i> mutant cells. The cells were untreated (UT) or stimulated with 250 nM thapsigargin (Tg) or 0.5 mM histidinol (His) for 24 hours. Note the enhanced response of the <i>CHOP</i>::<i>GFP</i> ISR reporter. (B) Bar diagram of the median ± S.D. of the reporter gene activity from experiments as shown in “A”. N = 3, *P = 0.0057 for Tg, *P = 0.037 for His, Unpaired t test. (C) Experimental design for tracking <i>EIF2B4</i>^<i>A392D</i> mutations. A fluorescent protein-marked sgRNA/Cas9 plasmid targeting <i>EIF2B4</i> and a wildtype or <i>EIF2B4</i>^<i>A392D</i> mutant repair template marked by a silent <i>Spe</i>I mutation were co-transfected into CHO-S21 cells. Transfected cells (selected by FACS), were treated with histidinol and divided into four bins (Bin #1 to #4) by level of <i>CHOP</i>::<i>GFP</i> expression. After recovery, genomic DNA was isolated from cells in each bin and the targeted region of <i>EIF2B4</i> was amplified by PCR and digested with <i>Spe</i>I to reveal frequency of targeting by either repair template. (D) PCR fragments digested with <i>Spe</i>I from genomic DNA of the indicated bins, visualized on an agarose gel. Shown is an image of a representative experiment reproduced twice. (E) Plot of the distribution of <i>Spe</i>I digested fragments in the four bins of transduced cells from the experiment in “D”. The band intensities of the digested fragments (reporting on recombination of the wildtype or mutant repair template) were normalized to total PCR product intensity and the distribution of the relative frequency of recombination in the different bins was plotted. Note the enrichment for recombination of the <i>EIF2B4</i>^<i>A392D</i> mutant repair template in the ISR^High bin.</p

Severe VWM mutant cells are unable to tolerate a second <i>EIF2S1</i>^<i>S51A</i> mutation.

<p>(A) Experimental design for tracking <i>EIF2S1</i>^<i>S51A</i> mutant cells. A fluorescent-tagged sgRNA/Cas9 plasmid targeting <i>EIF2S1</i> was co-transfected alongside wild type (WT) or <i>EIF2S1</i>^<i>S51A</i> (Mut) templates into CHO-S21 dual reporter cells. Following FACS selection for the transfected cells they were treated with 250 nM thapsigargin (Tg) for 24 hours and reporter expression was analyzed. (B) Flow cytometry analysis of reporter activity in untreated (UT) and thapsigargin-treated (Tg) CHO-S21 cells from the experiment outlined in “A”. Note the emergence of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated cells in the pool offered an <i>EIF2S1</i>^<i>S51A</i> repair template. (C) Flow cytometry analysis of reporter activity in untreated (UT) and thapsigargin-treated (Tg) parental CHO-S21 or indicated VWM mutant cells following targeting of the <i>EIF2S1</i> locus with an <i>EIF2S1</i>^<i>S51A</i> repair template (as described in “A”). Note the lack of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated putative <i>EIF2S1</i>^<i>S51A</i><i>; EIF2B4</i>^<i>A392D</i> or <i>EIF2S1</i>^<i>S51A</i><i>; EIF2B4</i>^<i>R484W</i> double mutant cells (lower right panel). (D) Percentage of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated putative <i>EIF2S1</i>^<i>S51A</i> mutant cells in the indicated population from experiments as in “C”. Shown are means ± S.D. N = 6 (Parent), 5 (<i>EIF2B4</i>^<i>A392D</i>), and 3 (<i>EIF2B4</i>^<i>R484W</i> and <i>EIF2B4</i>^<i>R468W</i>). *** P<0.001, ** P<0.01, n.s. not significant, One way ANOVA followed by Dunnett’s multiple comparisons test.</p

Stress-resistance of wildtype and VWM cells.

<p>(A) Schema of experiments to compare the effect of thapsigargin in parental CHO-S21 and VWM mutant cells. Cells were treated with thapsigargin (Tg; 250 nM) for the indicated time, washed free of compounds and allowed to recover before assay. W = WST-1 assay, P = Puromycin labeling. (B) Immunoblot of puromycinylated proteins following a brief pulse of puromycin, reporting on levels of translation under the indicated experimental conditions. Shown is a representative experiment reproduced four times. P and E indicate parental CHO-S21 and <i>EIF2B4</i>^<i>A392D</i> mutant cells, respectively. (C) Quantification of “B”. Signal intensities of puromycinylated proteins were normalized by eIF2α. Shown are means ± SEM of four independent experiments. (D) Cell viability measured by the WST-1 assay in the experiment described in “A”. Shown are the mean ± SEM of four replicates of a representative experiment repeated three (R484W, R468W) to six (A392D) times.</p