Figure 5. Genome-wide CRISPR screen reveals lysosomal stress as a mediator of PARP1 downregulation in aneuploid cells.

(A) FACS-based genome-wide CRISPR screen to find PARP1 regulators. (B) Validation of PARP1 antibody. (C) Enrichment and depletion of sgRNAs compared to unsorted samples. (D) Normalized sgPARP1 read counts in sorted vs unsorted samples. (E) Pathway analysis of 269 PARP1 activators. (F) PCA of lysosomal (left) and proteasomal (right) gene expression in near-diploid (blue) and aneuploid (red) hCEC clones. Group separation assessed by 10,000 permutations using Euclidean distances in PC1-PC2 space. (G) Heatmap of log2FC z-scores for KEGG lysosomal genes in aneuploid cells and Bafilomycin A1 (Baf A1)-treated cells. (H) Spearman correlation of lysosomal gene expression changes in aneuploid cells and Baf A1-treated RPE1 cells. (I) PARP1 mRNA levels (Log2) in Baf A1-treated cells. (J) Viability of hCECs pre-treated with Baf A1 (5 nM, 24h) followed by H₂O₂ (200 μM, 24 h). (K) Viability after H₂O₂ in hCECs ± Baf A1 pre-treatment or PARP1 overexpression. One-way ANOVA with Dunnetts’ test (D); with Tukey’s test (K). Unpaired t-test (I, J).