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. 2015 Aug 18;29(10):1440–1453. doi: 10.1210/me.2015-1102

Figure 1.

Figure 1.

Generation of SMAD4-null PDAC cell lines with inducible expression of SMAD4. A, pINDUCER-SMAD4-Puro construct. pINDUCER is a lentiviral system that drives constitutive expression of the inducible transactivator rtTA3 and a puromycin resistance gene by the EF1a promoter while placing SMAD4 under the control of the Dox+rtTA3 inducible TRE2 promoter. SMAD4 is expressed after doxycycline treatment. Stable clones of the pancreatic cancer cell lines BxPC3 and CFPAC1 were selected by puromycin. B, Addition of doxycycline induces SMAD4 protein expression. Western blotting was performed on 15 μg of total protein isolated from BxPC3-pINS4c5 cells after a 24-hour culture with or without 1-μg/mL doxycycline. Addition of doxycycline induced SMAD4 protein expression. C, Addition of doxycycline induces SMAD4 protein expression. Western blotting was performed on 30 μg of total protein isolated from CFPAC1-pINS4c3 cells after a 24-hour culture with or without 1-μg/mL doxycycline. Addition of doxycycline induced SMAD4 protein expression. D, SMAD4 expression restores TGF-β signaling in BxPC3. Expression of TGF-β-inducible gene PAI1 was compared between untreated BxPC3-pINS4c5, BxPC3-pINS4c5 treated for 24 hours with 1-μg/mL doxycycline, and BxPC3-pINS4c5 treated for 24 hours with 1-μg/mL dox + 1-μg/mL TGF-β inhibitor SB-505124. PAI1 expression levels were normalized to ACTB and compared with BxPC3-pINS4c5 without doxycycline treatment. Error bars are ±SEM for technical triplicates. Comparisons are Student's 2-tailed paired t test with ***, P ≤ .001; **, P ≤ .005; *, P ≤ .05.