Fig. 1: The expression of both RasGRP1 and IL-6 is regulated by let-7a. | Nature Communications

Fig. 1: The expression of both RasGRP1 and IL-6 is regulated by let-7a.

From: RasGRP1 promotes the acute inflammatory response and restricts inflammation-associated cancer cell growth

Fig. 1

a qPCR analysis of microRNA (miRNA) Let-7 isoform expression in peritoneal macrophages (means ± SEM, n = three independent experiments). b RNA immunoprecipitation (RIP) of the miRNA let-7a binding Il6 mRNA. (means ± SEM, n = three independent experiments). c ELISA (top) of IL-6 in the supernatants of HEK293 cells transfected with the indicated molecules for 24 h (means ± SEM, n = three independent experiments, One-way ANOVA Tukey test, ***p = 0.0001, ***p = 0.0001 in sequence). Immunoblot analysis (down) of IL-6 in lysates of HEK293 cells of (c) (the image shown is a representative of three independent experiments). d qPCR analysis of Il6 in peritoneal macrophages transfected with the indicated molecules (means ± SEM, n = three independent experiments, One-way ANOVA Tukey test). e ELISA of IL-6 in the supernatants of peritoneal macrophages treated as in (d) (means ± SEM, n = three independent experiments, One-way ANOVA Tukey test, **p = 0.002, **p = 0.003). f Luciferase reporter analysis of Il6 3’ UTR reporter gene activity in HEK293 cells treated with the let-7a mimics or a matched control (means ± SEM, n = three independent experiments, One-way ANOVA LSD test, **p = 0.002). g Microarray analysis of mouse peritoneal macrophages treated with 100 ng/ml LPS. h qPCR analysis of predicted of the miRNA let-7-targeted genes in mouse peritoneal macrophages (means ± SEM, n = three independent experiments). i Immunoblot analysis of RasGRP1 and β-actin in lysates of HEK293 cells transfected with the indicated molecules for 24 h. j Immunoblot analysis of RasGRP1 and β-actin in lysates of HEK293 cells transfected as described in (i), but with Rasgrp1 (CDS + 3’ UTR) replacing Rasgrp1 (CDS). k Luciferase reporter analysis of Rasgrp1 3’ UTR reporter gene activity in HEK293 cells (means ± SEM, n = three independent experiments, One-way ANOVA LSD test, ***p = 0.0001). The data shown in i and j are from one representative experiment of three independent experiments. ns, not significant; *p < 0.05, ** p < 0.01, ***p < 0.001.

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