Figure 2.
Figure 2. Truncating PPM1D mutations lead to decreased degradation of PPM1D. (A) Log2-fold enrichment of gRNAs (black dots) in Molm13 cells exposed to cytarabine treatment versus vehicle treatment of 12 days. The experiment was performed with biological triplicates, and the red line represents the locally weighted scatterplot smoothing (LOESS) of 0.1. gRNAs with a z score ≥ 3 are shown in green. Overlaid are the absolute number of somatic PPM1D frameshift and nonsense mutations (black bars) identified in the blood cells of 28 418 individuals as described in Figure 1A. (B) Whole-cell lysates from Molm13 cells overexpressing full -length PPM1D (Full-Length PPM1D) or truncated PPM1D (Truncated PPM1D) were collected at different time points following cycloheximide (50 μg/mL) treatment. Blots were probed with anti-PPM1D and anti-COXIV. (C) Whole-cell lysates before and after 4 hours of 10 μM MG132 treatment from Molm13 cells overexpressing full-length PPM1D (Full-Length PPM1D) or truncated PPM1D (Truncated PPM1D) cDNA. (D) Vector map of the degradation reporter vector. Different PPM1D cDNA constructs are cloned in-frame with EGFP, allowing for monitoring of PPM1D expression levels through EGFP expression. mCherry is expressed following an IRES sequence and provides an internal control for vector expression in each cell. (E) EGFP-to-mCherry ratio in Molm13 cells with overexpression of full-length PPM1D, truncated PPM1D or the C-terminal end of PPM1D. The EGFP-to-mCherry ratios are normalized to the expression level of full-length PPM1D, and provide an estimate for the level of degradation. Experiments were done using biological triplicates and data are shown as the means ± SD. Unpaired Student t tests were used to calculate the association between the different vectors and P values were corrected for multiple hypothesis testing. (F) EGFP-to-mCherry ratio in Molm13 p53−/− cells with overexpression of full-length PPM1D, truncated PPM1D or the C-terminal end of PPM1D. The EGFP-to-mCherry ratios are normalized to the expression level of full-length PPM1D. Unpaired Student t tests were used to calculate the association between the different vectors and P values were corrected for multiple hypothesis testing. (G) EGFP-to-mCherry ratio in Molm13 cells before and after exposure to MG132 (10 μM, 6 hours), normalized to pretreatment values. Paired Student t tests were used to compare between treatment groups. Values represent means ± SD of biological replicates. (H) Cell viability analysis in Molm13 control cells (control), Molm13 PPM1D-truncating mutant cells (PPM1D-mutant) and Molm13 cells with overexpression of wild-type PPM1D (WT overexpression). Cells were exposed to increasing concentrations of cytarabine for 72 hours. Experiments were performed with biological replicates and data are shown as the means ± SD. Nonlinear logistic regression analyses and a sum of squares F test were performed to compare the inhibitory response to cytarabine in Molm13 WT overexpression cells and Molm13 PPM1D-truncating mutant cells to the inhibitory response in Molm13 control cells.

Truncating PPM1D mutations lead to decreased degradation of PPM1D. (A) Log2-fold enrichment of gRNAs (black dots) in Molm13 cells exposed to cytarabine treatment versus vehicle treatment of 12 days. The experiment was performed with biological triplicates, and the red line represents the locally weighted scatterplot smoothing (LOESS) of 0.1. gRNAs with a z score ≥ 3 are shown in green. Overlaid are the absolute number of somatic PPM1D frameshift and nonsense mutations (black bars) identified in the blood cells of 28 418 individuals as described in Figure 1A. (B) Whole-cell lysates from Molm13 cells overexpressing full -length PPM1D (Full-Length PPM1D) or truncated PPM1D (Truncated PPM1D) were collected at different time points following cycloheximide (50 μg/mL) treatment. Blots were probed with anti-PPM1D and anti-COXIV. (C) Whole-cell lysates before and after 4 hours of 10 μM MG132 treatment from Molm13 cells overexpressing full-length PPM1D (Full-Length PPM1D) or truncated PPM1D (Truncated PPM1D) cDNA. (D) Vector map of the degradation reporter vector. Different PPM1D cDNA constructs are cloned in-frame with EGFP, allowing for monitoring of PPM1D expression levels through EGFP expression. mCherry is expressed following an IRES sequence and provides an internal control for vector expression in each cell. (E) EGFP-to-mCherry ratio in Molm13 cells with overexpression of full-length PPM1D, truncated PPM1D or the C-terminal end of PPM1D. The EGFP-to-mCherry ratios are normalized to the expression level of full-length PPM1D, and provide an estimate for the level of degradation. Experiments were done using biological triplicates and data are shown as the means ± SD. Unpaired Student t tests were used to calculate the association between the different vectors and P values were corrected for multiple hypothesis testing. (F) EGFP-to-mCherry ratio in Molm13 p53−/− cells with overexpression of full-length PPM1D, truncated PPM1D or the C-terminal end of PPM1D. The EGFP-to-mCherry ratios are normalized to the expression level of full-length PPM1D. Unpaired Student t tests were used to calculate the association between the different vectors and P values were corrected for multiple hypothesis testing. (G) EGFP-to-mCherry ratio in Molm13 cells before and after exposure to MG132 (10 μM, 6 hours), normalized to pretreatment values. Paired Student t tests were used to compare between treatment groups. Values represent means ± SD of biological replicates. (H) Cell viability analysis in Molm13 control cells (control), Molm13 PPM1D-truncating mutant cells (PPM1D-mutant) and Molm13 cells with overexpression of wild-type PPM1D (WT overexpression). Cells were exposed to increasing concentrations of cytarabine for 72 hours. Experiments were performed with biological replicates and data are shown as the means ± SD. Nonlinear logistic regression analyses and a sum of squares F test were performed to compare the inhibitory response to cytarabine in Molm13 WT overexpression cells and Molm13 PPM1D-truncating mutant cells to the inhibitory response in Molm13 control cells.

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