Fig. 5

Inhibition of Notch signal reversed stem cell phenotypes induced by KLF10 depletion. A (Upper panels) representative immunoblots of KLF10 and Notch signal pathway molecules as indicated in Panc-1-pLKO or Panc-1-pLKO-shKLF10 cells without or with Notch-3 (left panel) or Notch-4 (right panel) genes silencing. β-Actin was used as internal control. (Lower panels) the bar graphs were mean ± SE from cumulated data of three independent experiments. Levels of signal molecule expression in Panc-1-pLKO (yellow) or Panc-1-pLKO-shKLF10 (blue) cells without (filled) or with Notch 3/4 (shN3/4, diagonal stripe) receptor mRNA silencing were measured. *p < 0.05. B Sphere formation of Panc-1-pLKO (yellow) or Panc-1-pLKO-shKLF10 (blue) cells without (filled) or with Notch 3 (shN3, diagonal stripe) or Notch 4 (shN4, diagonal check) receptor mRNA silencing. The bar graphs were mean ± SE from cumulated data of three independent experiments. *p < 0.05 and **p < 0.01, respectively. C (Left panels) representative flow cytometry of CD24 and c-Met on Panc-1-pLKO (upper panels) and Panc-1-pLKO-shKLF10 (lower panels) cells without or with Notch-3/4 mRNA silencing as indicated. (right panels) Expression of CD24 (left panel) and c-Met (right panel) on Panc-1-pLKO (yellow) or Panc-1-pLKO-shKLF10 (blue) cells without (filled) or with Notch-3 (sN3, diagonal stripe) or Notch-4 (sN4, diagonal check) mRNA silencing. The bar graphs were mean ± SE from cumulated data of three independent experiments. **p < 0.01 and ***p < 0.005, respectively. D (Left panel) representative immunoblots of KLF10 and Notch signal molecules in Panc-1-pLKO or Panc-1-pLKO-shKLF10 cells without or with 20 µM DAPT treatment. β-Actin was used as internal control. (Right panel) the bar graphs were mean ± SE from cumulated data of three independent experiments. Levels of signal molecule expression in Panc-1-pLKO (yellow) or Panc-1-pLKO-shKLF10 (blue) cells without (filled) or with DAPT (diagonal stripe) treatment were measured. *p < 0.05. E Sphere formation of Panc-1-pLKO (yellow) or Panc-1-pLKO- shKLF10 (blue) cells without (filled) or with (diagonal stripe) 20 µM DAPT treatment. The bar graphs were mean ± SE from cumulated data of three independent experiments. *p < 0.05. F (Upper panels) representative flow cytometry of CD24 and c-Met on Panc-1-pLKO (left panels) and Panc-1-pLKO-shKLF10 (right panels) cells without or with DAPT treatment as indicated. (Lower panels) expression of CD24 (left panel) and c-Met (right panel) on Panc-1-pLKO (yellow) or Panc-1-pLKO-shKLF10 (blue) cells without (filled) or with (diagonal stripe) 20 µM DAPT treatment. The bar graphs were mean ± SE from cumulated data of three independent experiments. *p < 0.05. G Representative IVIS images of mice at 2–6 weeks after implanting orthotopically with Panc-1-pLKO or Panc-1-pLKO-shKLF10 cells without or with DAPT 15 mg/kg intra-peritoneally for 3 weeks as described in “Materials and methods” (left panel). Representative photos of tumors resected from mice experiments mentioned above (right panel). H Cumulated IVIS signal of at least six mice in each experimental group that were implanted with Panc-1-pLKO (yellow) and Panc-1-pLKO-shKLF10 (blue) without (solid) or with (dashed line) DAPT treatment for 3 weeks as indicated. Each point represents mean ± SE. *p < 0.05. I Representative H&E (upper panel) and IHC staining, as described in “Materials and methods”, of KLF10, NICD-3 and -4, as indicated, in tumors resected from mice experiments of G