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

Figure 2.

Figure 2.

Restoration of TGF-β signaling induces G1 arrest and senescence in PDAC cell line BxPC3. A, SMAD4 expression in BxPC3 induces G1 arrest 24 hours after doxycycline addition. A total of 1-μg/mL doxycycline was added to experimental wells. Twenty-four hours after doxycycline treatment, cells were fixed with 70% EtOH and stained with 40-μg/mL PI for flow cytometry to stratify cells by DNA content. FlowJo's Cell Cycle module with Watson model was used to determine the cell cycle phase distribution of each sample based on the PI staining. The increase in the G1 phase population after doxycycline treatment indicates G1 arrest. B, SMAD4-induced G1 arrest is dependent on TGF-β signaling. SB-505124, an Activin receptor-like kinase 4/5/7 inhibitor, was added to experimental wells at 1 μg/mL. After 2 hours, 1-μg/mL doxycycline was added to experimental wells. Twenty-four hours after doxycycline treatment, cells were fixed with 70% EtOH and stained with 40-μg/mL PI for flow cytometry to stratify cells by DNA content. FlowJo's Cell Cycle module with Watson model was used to determine the cell cycle phase distribution of each sample based on the PI staining. SB-505124 pretreatment eliminated the G1 arrest effect of SMAD4 expression seen in A. C, SMAD4 expression induces senescence in BxPC3-pINS4c5. BxPC3-pINS4c5 cells in the experimental condition were treated with 1-μg/mL doxycycline. Seventy-two hours later, the samples were stained using Senescence β-galactosidase Staining kit (Cell Signaling). The presence of blue-stained cells indicates senescence. D, SMAD4 expression induces senescence-associated repression of LMNB1 in BxPC3-pINS4c5. Expression of LMNB1, a gene repressed in senescent cells, was compared by RT-qPCR between untreated BxPC3-pINS4c5 and BxPC3-pINS4c5 treated for 24 hours with 1-μg/mL doxycycline. LMNB1 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.