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. 2013 Feb 8;123(3):1348–1358. doi: 10.1172/JCI65416

Figure 3. TGF-β increases the CSC population in a SMAD4-dependent manner.

Figure 3

(A) SUM159 and BT549 cells were preincubated with 1 to 5 μM LY2157299 for 24 hours, followed by treatment with 2.5 ng/ml TGF-β1 for 2 hours in media supplemented with 0.5% FBS. Cell lysates were prepared and subjected to immunoblot analysis with total SMAD2/3 and P-SMAD2 antibodies. (B) SUM159 and BT549 cells were treated with 2.5 ng/ml TGF-β1 with or without 5 μM LY2157299 for 6 days. TGF-β1 and inhibitor were replenished every 3 days. ALDEFLUOR assay was performed in SUM159 cells as described in Methods (n = 3; *P < 0.01). BT549 cells were stained with CD44 and PROCR antibodies, followed by FACS analysis (n = 3; #P < 0.002). Error bars indicate SEM. (C) SUM159 and BT549 cells were seeded in low-adherent dishes and treated with 2.5 ng/ml TGF-β1 with or without 5 μM LY2157299 for 6 days. TGF-β and inhibitor were replenished every 3 days. Mammosphere number was calculated using a GelCount reader and software. Each bar represents the mean number ± SEM (*P < 0.02). (D) SUM159 and BT549 cells were transfected with nontargeting control (CTL) or 2 different SMAD4 siRNA oligonucleotides for 72 hours; lysates of these cells were separated by SDS-PAGE and subjected to immunoblot analysis with SMAD4 and actin antibodies. (E) Control and SMAD4 siRNA-transfected SUM159 and BT549 cells were cultured as mammospheres in the presence or absence of 2.5 ng/ml TGF-β1. Mammosphere number was calculated using a Gel Count reader and software after 6 days. Each bar represents the mammosphere number ± SEM (n = 3; *P < 0.04).