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. Author manuscript; available in PMC: 2010 Nov 13.
Published in final edited form as: Cell. 2009 Oct 29;139(4):693–706. doi: 10.1016/j.cell.2009.10.014

Figure 4.

Figure 4

Let-7a regulates IL6 expression during transformation

(A) Let-7a binding site in 3′UTR of IL6 with sequence complementarity and phylogenic conservation (yellow) of IL6 target sequence indicated.

(B) Luciferase assay using a reporter containing the 3′UTR of IL6 (wt or mutant in let-7a binding site) 24h after transfection with let-7a or a scrambled miR control.

(C) Physical interaction between let-7a and IL6 mRNA in the context of the RISC complex. IL6 RNA levels in HA-Ago1 immunoprecipitates in HEK-293 cells co-transfected with a plasmid expressing HA-Ago1 and let-7a or scrambled microRNA control.

(D) IL6 RNA levels after treatment for 24h with antisense-let-7a (100 nM) or Lin28B.

(E) Phosphorylation of STAT3-Y705 ELISA assay; mean ± SD) in untreated and TAM-treated (36h) cells in addition to treatment with increasing concentrations (50, 80, 100 nM) of let-7a and siRNA against Lin28B (80 nM).

(F) VEGF and IL6 production (ELISA assay; mean ± SD) in TAM-treated cells before and after transfection (100 nM) of the indicated let-7 microRNAs.

(G) Western blot analysis of RAS protein expression in 36h TAM-treated cells after let-7a (100 nM) overexpression; GAPDH levels were used as a loading control.

(H) IL6 production (ELISA assay during transformation after transfection with negative control siRNA or siRNA against Ras or let-7a (100 nM).

(I) Soft agar colony assay in TAM-treated cells after treatment with 2 μg/ml of monoclonal antibody against IL6 (Ab-IL6) and IgG isotype antibody (Ab-IgG) or siRNA negative control and siRNA against Ras (100 nM).