Fig. 5: NAD(P)H:quinone oxidoreductase 1 (NQO1) is a critical factor for CBK77-mediated UPS impairment.

A Schematic drawing depicting the pooled CRISPR–Cas9 screening paradigm. MelJuSo cells expressing Cas9 were infected with a lentiviral sgRNA library (four sgRNAs per gene). Cells were treated with DMSO (0.25%) or CBK77 25 µM three times during a 5-day period. The resulting cell populations were harvested and subjected to barcode sequencing and analysis. Two technical replicates were performed. B Dose-response experiments performed with MelJuSo Ub-YFP cells. Cell viability was assessed after 72 h. Data are represented as mean ± SD of three independent experiments. Nonlinear curve fitting is depicted in red. The half-maximal inhibitory concentration (IC₅₀) and slope of the curve are used to calculate the theoretical IC₉₀ concentration. C Scatter plot of the average gene log-fold change (LFC) in CBK77-treated cells versus control (CTRL)-treated cells in the two replicates as calculated using MaGeCK [17]. Each dot represents a gene and is colored from lower (blue) to higher (red) LFC values. The names of genes with an average LFC > 1.5 are shown. D MelJuSo Ub-YFP cells were depleted of NQO1 for 48 h with the indicated siRNAs (10 nM) and then treated for 24 h with CBK77 (5 µM). Nontargeting siRNA (siCTRL) was used as control. After the treatment, nuclei were counterstained with Hoechst and cells imaged live with a widefield automated microscope. Frequency and distribution of the nuclear YFP intensity per cell are shown as violin plots from one experiment (of two independent experiments). Black lines within each distribution represent the median; white lines represent the upper and lower interquartile range limits. The knockdown efficiencies are shown in the blot below. E MelJuSo Ub-YFP cells were co-treated with either DMSO, CBK77, CBK07 (10 µM) or bortezomib (PI proteasome inhibitor, 25 nM) with or without the NQO1 inhibitor ES936 (100 nM) for 6 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Representative blots from one of three independent experiments are shown. F MelJuSo Ub-YFP cells were treated with CBK77 (10 µM) alone or in combination with the NQO1 inhibitor ES936 at the indicated concentrations for 24 h. After the treatment, cells were fixed and nuclei counterstained with Hoechst. Cells were imaged with an automated widefield microscope. The percentage of YFP-positive cells are shown as mean of three independent experiments. Error bars depict the SEM. ns nonsignificant, * adjusted p ≤ 0.05 (one-way ANOVA with Dunnett’s multiple comparisons test). G The cell count from F was obtained based on the nuclei staining. Data are shown as mean of three independent experiments. Error bars depict the SEM. ns nonsignificant, * adjusted p ≤ 0.05, ** adjusted p ≤ 0.01 (one-way ANOVA with Dunnett’s multiple comparisons test).