Figure 6. TGF-β positively regulates the production of CCL22 and Treg cell migration via repression of miR-34a in PVTT-1 cells.

(A) Time course of TGF-β-induced CCL22 production detected in the culture media of PVTT-1 cells. CCL22 was detected by Western blot and γ-tubulin in corresponding cell lysate was used as a loading control.

(B) PVTT-1 cells were pretreated with the control solvent (DMSO) or SB431542 (SB) at 20 µM for 30 min and then treated with TGF-β1 for 12 hrs. The expression of miR-34a (left panel) and of CCL22 mRNA (right panel) were measured by qRT-PCR and normalized to U6 or GAPDH, respectively (N=3, Mean±s.d.). **p<0.01 (Student t-test).

(C) PVTT-1 cells were pretreated with various doses of pre-miR-34a mimetic or control oligos for 24 hrs, before TGF-β1 treatment for another 12 hrs as indicated. The level of CCL22 mRNA was measured by qRT-PCR and normalized to GAPDH (left panel). CCL22 protein was detected by Western blot and γ-tubulin was used as a loading control (right panel).

(D) The same transwell assay as in Figure 5A was conducted with supernatants from PVTT-1 cells first transfected with pre-miR-34a or control oligos, and 24 hrs later treated with TGF-β1 for another 24 hrs. (N=3, mean±s.d.). **p<0.01 (Student t-test).

All error bars indicate mean±SD.