Figure 4.

Up-regulation of CDKN1A/p21 is necessary for G₁ arrest and senescence effects of SMAD4 expression in PDAC cell line BxPC3. A, SMAD4-induced G₁ arrest in BxPC3 is dependent on up-regulation of CDKN1A/p21. BxPC3-pINSc5 cells were transfected with pooled CDKN1A-siRNA, pooled NT-siRNA, or a mock treatment. Twenty-four hours after transfection, experimental cells were treated with 1-μg/mL doxycycline. Twenty-four hours after doxycycline treatment, cells were fixed with 70% EtOH and stained with 40-μg/mL PI for flow cytometry to determine cell cycle status. FlowJo's Cell Cycle module with Watson model was used to determine the cell cycle phase distribution of each sample. CDKN1A/p21-siRNA eliminated the G₁ arrest effect of SMAD4 expression. B, SMAD4-induced senescence in BxPC3 is dependent on up-regulation of CDKN1A/p21. BxPC3-pINSc5 cells were transfected with pooled CDKN1A-siRNA, pooled NT-siRNA, or mock treatment. Twenty-four hours after transfection, experimental cells were treated with 1-μg/mL doxycycline. Twenty-four hours after doxycycline treatment, mRNA was isolated. LMNB1 expression levels were assessed by RT-qPCR, normalized to ACTB, and compared with BxPC3-pINS4c5 mock transfected without doxycycline treatment. CDKN1A/p21-siRNA eliminated the senescence-associate repression of LMNB1 by SMAD4 expression. Error bars are ±SEM for technical triplicates. Comparisons are Student's 2-tailed paired t test with **, P ≤ .001; *, P ≤ .005.