Fig 2: Inactivation by MI2321 alters the structure of NQO1. (A) Schematic representation of the three conformations of NQO1 and their immunoreactivity towards the redox-specific anti-NQO1 C-terminus antibody. (B) 16HBE cells were pretreated with DMSO, MI2321 (1 µM) or MI3190 (1 µM) for 30 min followed by exposure to 2 mM H2O2 for 2 h. NQO1 was then immunoprecipitated from lysates (1 mg) using the anti-NQO1 C-terminus antibody. ß-Lapachone (ß-Lap), 10 µM × 2 h was included as a positive control for oxidized NQO1. (C) Purified recombinant human NQO1 (0.2 µg) was incubated in the absence and presence of NADH (200 µM) for 15 min then hydrogen peroxide (5 mM) was added for an additional 1 h. Following incubations, catalase (800 µg/ml) was added to each tube and NQO1 was immunoprecipitated using the anti-NQO1 C-terminus antibody. (D) Migration of purified rhNQO1 following inactivation by MI2321. Purified rhNQO1 (2.5 µg) was incubated with 500 µM NADH and 50 µM MI2321 for 30 min then analyzed by non-denaturing PAGE.

Fig 3: Inactivation of NQO1 results in a temporal increase in acetyl a-tubulin levels in 16HBE cells. (A) Acetyl a-tubulin levels were measured in 16HBE cells after treatment with MI2321 (1 µM) for the indicated times. (B) NQO1 catalytic activity was measured in 16HBE cell lysates obtained from cells treated with DMSO or MI2321 (1 µM) for the indicated times.

Fig 4: 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).

Fig 5: Inactivation of NQO1 by MI2321 results in lower levels of immunostaining for NQO1 and higher levels of perinuclear acetyl a-tubulin in intact cells. 16HBE cells (top panels) were treated with DMSO or MI2321 (1 µM) for 2 h while 3T3-L1 fibroblasts (bottom panels) were treated with DMSO or MI2321 (1 µM) for 30 min. Following treatments the cells were fixed then immunostained for NQO1 and acetyl a-tubulin. NQO1 immunostaining was performed using the redox-specific anti-NQO1 A180 antibody.