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. 2022 Apr 14;12(4):64. doi: 10.1038/s41408-022-00663-z

Fig. 4. Identification of mutations on V49 and neighboring residues of PDE6D hydrophobic domain as essential for cellular resistance to DW0254.

Fig. 4

A Percentage of live P12-ICHIKAWA cells by DAPI staining after transduction with either PDE6D library or empty vector control treated for two weeks with 2 μM of DW0254 or DMSO, data represent mean ± SD of three technical replicates, ***P ≤ 0.001. B Changes in barcoded sgRNAs of untreated PDE6D library cells 14 days after transduction. The cDNA position (in bp) is shown on the X-axis. The fold-change in CRISPR score is shown on the Y-axis. Negative and positive controls are shown in green and red dots, respectively. Negative controls used were non targeting sgRNAs and positive controls targeting essential genes, including PCNA, CDK1, CDK9, RPA3, BRD4, MYC, and RPS20. C Changes in barcoded sgRNAs of PDE6D library cells treated for 14 days with 2 μM of DW0254 versus 14 days of DMSO. Dotted line on panels (B) and (C) represents a 20-fold change on CRISPR score. D DW0254 dose response curves showing % of viable PDE6D library cells or controls untreated or treated with DW0254 at 2 µM for 14 days. E Deltarasin dose response curves showing % of viable empty vector transduced cells, untreated PDE6D library cells and PDE6D library cells treated with DW0254 at 2 µM for 14 days. F Cell growth curves for P12-ICHIKAWA cells expressing Cas9 only or Cas9 and sgRNA144, treated with 2.5 μM of DW0254 for 21 days. G DW0254 dose response curves showing % of viable empty vector transduced cells, untreated sgRNA144 transduced cells, and sgRNA144 cells treated with DW0254 for 21, 50, and 80 days. H Deltarasin dose response curves showing % of viable untreated sgRNA144 transduced cells and controls, and sgRNA144 cells treated with DW0254 for 21, 50, and 80 days; For panels (D), (E), (G) and (H): data represent mean ± SD of two independent experiments with N = 3 samples for each condition. I DW0254 dose response curves showing % of viable cells transduced with empty vector and two single cell clones of sgRNA144 transduced cells. J Deltarasin dose response curves showing % of viable empty vector and two single cell clones of sgRNA144 transduced cells; For panels (I) and (J) data represent mean ± SD of two independent experiments with N = 4 samples for each condition.