Fig. 4: Depletion of USP25 leads to reduced patient-derived organoid formation, viability and attenuated PDAC tumor growth in vivo.

a Representative images of shUsp25 silencing in KPCY organoids. Scale bar is 800 µm. b KPCY organoid viability displayed as % relative to shYFP controls (n = 4 biologically independent samples). c Western blot of endogenous Usp25 in KPCY organoids. d Representative images of shUSP25 silencing in PDO lines. Scale bar is 800 µm. e PDO viability displayed as % relative to shNT controls (n = 4 biologically independent samples). f Representative images of PDO lines incubated with caspase-3 activity probe. Scale bar is 800 µm. g Quantification of caspase-3 signal in (f), shown as mean fluorescent intensity (MFI). Each symbol represents a different shRNA targeting either USP25 or NT control, averaged from biologically independent experiments (n = 3). h Representative images of the PDO lines (K4T), showing empty vector (EV) control and CRISPR/Cas9-mediated USP25 knock-out cells. Each USP25^−/− clones are described with single guide (sg) targeting exon shown. Scale bar is 400 µm. i Organoid viability displayed as % relative to the EV. j Western blot of endogenous USP25. k Representative images of PDO parental and USP25^−/− clones. Scale bar is 400 µm. l PDOX tumor volumes at end point. m Histological analysis of PDOX at end point. Scale bar is 1000 µm, inserts are 100 µm and 50 µm. Bar graphs in (b, e, g, i, l) are displayed as mean ± SD, and each colored circle in (b, e, i) represents a different organoid line, derived from an independent animal or patient. Statistical significance in (g, l) was determined by two-tailed Student’s t test, and in (b, e, i) by One-way ANOVA with Dunnett post-hoc test. Source data are provided as a Source Date file.