Figure 3. Targeted sublibrary senolytic Death-seq screens highlight importance of SMAC.
(A) Validation of DIABLO/SMAC sgRNA knockouts compared to control gRNA viability after treatment with vehicle or 1 μM ABT-263 for 3 d in Doxo-induced senescent IMR-90s. Data are representative of two independent experiments performed in triplicate and are presented as mean ± s.e.m. One-way ANOVA with Dunnett’s post-hoc test relative to control sgRNA-treated cells in the corresponding drug treatment, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
(B) Schematic of targeted custom sublibrary screens. The targeted custom library was infected using lentivirus into Cas9-expressing IMR-90 normal lung fibroblasts. The IMR-90s expressing the targeted sublibrary were then induced to senesce with Doxo and treated with 1 μM ABT-263, 2 μM birinapant, or 10 μM ABT-199 for 24 h. A targeted custom sublibrary screen was also performed with H2O2-induced senescent IMR-90s and 1 μM ABT-263 treatment. Like the genome-wide screen, Death-seq was used to compare the dying cell populations directly with the live attached cells. The screens were performed in duplicate.
(C) Volcano plot of the effects and confidence scores of all the genes in the targeted custom CRISPR screen in Doxo-induced senescent IMR-90 cells treated with 1 μM ABT-263 for 24 h. Labelled are the 80 genes passing 10% FDR for inhibiting (red) or promoting (blue) cell death by ABT-263 when knocked out.
(D) Correlation of combo casTLE confidence scores of the ABT-263 and birinapant screens in Doxo-induced senescent IMR-90s for all genes in the targeted custom sublibrary. R-squared value is from a linear regression model. Labelled in light green, purple, and orange are hits passing 10% FDR only in ABT-263 screen, only in birinapant screen, or in both screens, respectively.
(E) Correlation of combo casTLE confidence scores of the ABT-263 and ABT-199 screens in Doxo-induced senescent IMR-90s for all genes in the targeted custom sublibrary. R-squared value is from a linear regression model. Labelled in light green, pink, and dark green are hits passing 10% FDR only in ABT-263 screen, only in ABT-199 screen, or in both screens, respectively.
(F) Schematic of targeted custom sublibrary ABT-263 screen in Doxo-SEN WI-38 normal lung fibroblasts run head-to-head against Doxo-SEN IMR-90s. The screens were performed in duplicate.
(G) Correlation of combo casTLE confidence scores of the Doxo-induced senescent IMR-90s and Doxo-induced senescent WI-38s ABT-263 screens for all genes in the targeted custom sublibrary. R-squared value is from a linear regression model. Labelled in grey, dark brown, and light brown are hits passing 10% FDR only in the IMR-90 screen, only in the WI-38 screen, or in both screens, respectively.
See also Figure S2 and the results, raw sequencing counts, and composition of the targeted custom sublibrary from screens in Tables S1, S2, and S3 respectively.