Fig. 2: Usp15 functions as PDAC tumor suppressor.

A Tumor-free survival of Pdx1-Cre;LSL-Kras^G12D;LSL-Cas9-GFP mice injected with CRISPR AAV-sgRNAs targeting the indicated gene or non-targeting control sgRNA (sgCtrl, n = 6). Two independent sgRNAs were used (sgUsp15_1 n = 9, sgUsp15_2 n = 9). Log-Rank test (Mantel-Cox). B Representative H&E images showing multifocal PanINs in sgCtrl transduced pancreas and PADC tumors in sgUsp15 transduced pancreas. Scale bar 100 µm. C Tumor-free survival of Pdx1-Cre;LSL-Kras^G12D mice with the indicated Usp15 genotype where ‘+’ indicates the wildtype allele and ‘Δ’ indicates a conditionally deleted allele. KrasG12D +/- Usp15 +/+ (n = 12); KrasG12D +/- Usp15 +/- (n = 11); KrasG12D +/- Usp15 -/- (n = 13). Log-Rank test (Mantel-Cox). D Representative H&E images of mice with the indicated genotype showing multifocal PanINs and PADC tumors. Scale bar 100 µm. E Cell proliferation curves of KC cells transduced with the indicated sgRNA obtained using the IncuCyte live-cell imaging. Cells were grown for five days and data are expressed as cell confluence percentage (%; mean ± SD, n = 3 independent experiments, Two-way ANOVA (sgUsp15_1 p = 3.43e-8: sgUsp15_2 p = 4.89e-9), Dunnett’s multiple comparison. F Cell proliferation curves of KC cells expressing ubiquitin variants inhibiting Usp15 (Ubv15.1a and Ubv15.1/d) or wildtype ubiquitin (Ub^wt) as control. (%; mean ± SD, n = 3 independent experiments, Two-way ANOVA (Ubv15.1a p = 2.74e-6: Ubv15.1/d p = 2.06e-7), Dunnett’s multiple comparison. G Tumor-free survival of NSG (NOD Scid Gamma) mice after orthotopic injection sgCtrl (n = 5) or sgUsp15_1/2 (n = 5; n = 5) KC cells. Two independent sgRNAs were used. Log-Rank test (Mantel-Cox). H Dose-response curves for KPC sgCtrl or sgUsp15 cells treated with the indicated concentration of Olaparib (mean ± SD, n = 3 independent experiments). Two-way ANOVA (sgUsp15_1 p = 0.0349: sgUsp15_2 p = 0.0431), Dunnett’s multiple comparison.