Fig. 2

miR-221 prompts the protein expression of c-Rel and its recruitment to the CTGF promoter region. c-Rel protein expression in CAFs (a), MDA-MB 231 (b) and SkBr3 (c) cells transfected for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i). β-actin serves as a loading control. Below panels show densitometric analysis of the blots normalized to the loading controls. d Putative NF-kB binding site (capital letters in the rectangle) located within the CTGF promoter sequence (− 625 bp to + 62 bp). The transcriptional start site is indicated as + 1. The position of primers used for ChIP-qPCR analyses is underlined. Recruitment of c-Rel to the NF-kB binding site within the CTGF promoter sequence in CAFs (e), MDA-MB 231 (f) and SkBr3 (g) cells transfected for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i). Data were normalized to the Input and reported as fold changes respect to IgG. h Luciferase activity of the CTGF reporter gene in CAFs, MDA-MB 231 and SkBr3 cells transfected for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i). i Luciferase activity of the CTGF reporter gene in CAFs, MDA-MB 231 and SkBr3 cells transfected for 8 h with shRNA or shRel and then transfected for 48 h with 25 nM miR-221. The luciferase activity was normalized to the internal transfection control; values of cells receiving scrambled controls were set as 1-fold induction upon which the activity obtained with the indicated effectors was calculated. Each column represents the mean ± SD of three independent experiments performed in triplicate. (**) indicates p < 0.01