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. 2016 Aug 16;7(38):61262–61272. doi: 10.18632/oncotarget.11311

Figure 5. Activation of SMAD signaling is required for the down-regulation of PTEN by TGF-β1.

Figure 5

(A) KLE and HEC-50 cells were treated without (C) or with 10 ng/mL of TGF-β1 (T) for 30 and 60 minutes. Western blot was used to examine the levels of phosphorylated SMAD2 (p-SMAD2) or SMAD3 (p-SMAD3) in relation to their total levels from the same membrane (SMAD2 and SMAD3, respectively). (B) KLE and HEC-50 cells were transfected for 48 h with 20 nM control siRNA (si-Ctrl) or SMAD4 siRNA (si-SMAD4) and then treated without (Ctrl) or with 10 ng/mL of TGF-β1 for 6 or 24 h. PTEN mRNA levels were examined by RT-qPCR (normalized to GAPDH mRNA levels). (C) KLE and HEC-50 cells were transfected for 48 h with 20 nM control or SMAD4 siRNA, treated for a further 24 h with or without 10 ng/mL of TGF-β1, and PTEN protein levels were examined by Western blot (normalized to α-tubulin protein levels). Results are expressed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05).