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. 2019 Jan 25;169(1):108–121. doi: 10.1093/toxsci/kfz024

Figure 4.

Figure 4.

Disruption of Sec biosynthesis and utilization decreases cellular susceptibility to ATO/AsIII. A, Overview of the Sec biosynthesis pathway and incorporation into selenoproteins. B, Validation of the increased resistance to ATO upon disruption of genes involved in Sec biosynthesis and incorporation into proteins (obtained from secondary screening). Dots represent the different sgRNAs targeting each gene where the relative abundance of each sgRNA in the treated pools is divided by that in the control pools to calculate fold changes. The dotted line (y = 1) represents the baseline abundance of each sgRNA sequence in control pools. Data are represented as mean (n = 3). Only guide sequences displaying differential abundance at FDR < 0.001 are shown. Lines represent median fold enrichment of all guides corresponding to each gene. C, PSTK inactivation results in increased sensitivity to ATO. Cells were transduced with CRISPR/sgRNA vectors targeting PSTK or nontargeting control and treated with multiple doses of ATO for 96 h. Cell viability in each pool is shown as percentage of vehicle-treated control. Data are represented as mean of 3 independent experiments ± SD (n = 3). D, Relative enrichment (Fold Change > 1) of each of the guide sequences targeting TXNDC17 in ATO-treated compared to control pools. Fold changes for each guide sequence are calculated by dividing the normalized abundance of the sequence in ATO-treated pools by that in control pools. The dotted line (y = 1) in represents the baseline abundance of each guide sequence in control pools. Data are represented as mean (n = 3). All the guide sequences shown display higher abundance in ATO at FDR < 0.001. E, Selenium protection against ATO toxicity. K562 cells were pretreated with sodium selenite (10 μM) and ATO cytotoxicity was evaluated in pretreated and control cells at 48 h by CellTiter Glo cell viability assay. Cell viability is represented as percentage of vehicle-treated control. Data are represented as mean of 3 independent experiments ± SD. Statistical significance was determined by Student’s t-test, where *p < .05, **p < .01, ***p < .001.