Figure 6. Suppression of NOX4 by shRNA or DPI inhibits PDAC growth in vitro and in vivo.

(a,b) Growth curves and clonogenic growth were measured in NOX4-silenced HPNE/Kras^G12V/shp16 cells. (c) Weights of PDAC tumours removed on day 60 from mice (N=8) injected orthotopically with NOX4-silenced and control HPNE/Kras^G12V/shp16 cells. (d) The nude mice were inoculated subcutaneously with indicated cells (N=5). The tumour sizes were measured throughout the experiment to evaluate NOX4 knockdown effects. (e) Tumour weight derived from indicated group was measured. (f) Photograph and comparison of excised tumour size. (g) Sizes and weights of PDAC tumours (N=10) removed on day 42 from mice injected orthotopically with NOX4-silenced AsPc-1 cells or control AsPc-1 cells treated with NOX inhibitor DPI (1.5 mg kg⁻¹ per mouse, i.p., 5 days per week). (h) Paraffin-embedded tumour sections were stained with H&E (P: PDAC; S: Spleen) or anti-Ki67 antibody (Scale bars, 100 μm); apoptotic cells were visualized by TUNEL staining (green) and counterstained with DAPI (blue) (Scale bars, 10 μm). Quantification of proliferation index and apoptotic index in PDAC tumours was shown. (i) Sizes and weights of tumour tissues (N=5) removed on day 42 from mice injected orthotopically with AsPc-1/i-shNOX4 cells. On: mice were fed with doxy-containing water from 2 weeks after inoculation, and continued for 4 weeks. **P<0.01 for indicated comparison (Student unpaired t-test). (j) Kaplan–Meier overall survival analysis for mice treated with DPI as indicated (Kaplan–Meier analysis with the log-rank test). On: mice were fed with doxy-containing water from 3 weeks of age. DPI: mice were treated with DPI (1.5 mg kg⁻¹ per mouse, i.p., 5 days per week) from 8 weeks of age. Data in a,b are presented as mean±s.d. (n=3). Data in c–e,g,i are representative of two independent experiments and presented as mean±s.d. **P<0.01 for indicated comparison (one-way analysis of variance (ANOVA) with the Newman Keul's multiple comparison test).