Fig. 3: The 3’ UTR of Rasgrp1 increases IL-6 protein levels by sponging let-7a. | Nature Communications

Fig. 3: The 3’ UTR of Rasgrp1 increases IL-6 protein levels by sponging let-7a.

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

Fig. 3

a ELISA of IL-6 in HEK293 cells transfected with plasmids expressing the CDS and/or 3’ UTR of Rasgrp1 and Il6 and transfected with let-7a mimics for 24 h (means ± SEM, One-way ANOVA Tukey test, ***p = 0.0001, ***p = 0.0001 in sequence, n = three independent experiments). b RNA immunoprecipitation (RIP) analysis of the miRNA let-7a binding Il6 mRNA (means ± SEM, One-way ANOVA LSD test, **p = 0.002, ***p = 0.0001 in sequence, n = three independent experiments). c Schematic diagram showing the sequence of a sponge RNA. d qPCR of Il6 in peritoneal macrophages transfected with indicated molecular and treated 24 h later with 100 ng/ml lipopolysaccharide (LPS) for 6 h (means ± SEM, n = three independent experiments, One-way ANOVA Tukey test). e ELISA of IL-6 in the supernatants of peritoneal macrophages treated as described in (d) (means ± SEM, One-way ANOVA Tukey test, **p = 0.002, ***p = 0.0001, ***p = 0.0001 in sequence, n = 3 independent experiments). f Representative confocal microscopic image showing peritoneal macrophages cotransfected with indicated molecular (Scale bars represent 20 μm, the image shown is a representative of three independent experiments). g RIP analysis of the combination of let-7a and Il6 mRNA. (means ± SEM, One-way ANOVA Tukey test, ***p = 0.0001, ***p = 0.0001 in sequence, n = three independent experiments) h DNA sequencing of PCR products confirmed the targeted mutation in the RAW264.7 macrophage clone. i qPCR of Il6 in the RAW264.7 macrophage clones KO-1# or KO-2# (means ± SEM, n = three independent experiments, One-way ANOVA LSD test). j ELISA of IL-6 in the supernatants of the RAW264.7 macrophages treated as described in (i) (means ± SEM, One-way ANOVA LSD test, ***p = 0.0001, ***p = 0.0001, ***p = 0.0001, ***p = 0.0001, ***p = 0.0001, ***p = 0.0001 in sequence, n = three independent experiments). k ELISA of IL-6 in the supernatants of the RAW264.7 macrophage clone KO-1# transfected and treated with the indicated molecular (means ± SEM, One-way ANOVA Tukey test, ***p = 0.0001, ***p = 0.0001, ***p = 0.0001, ***p = 0.0001, **p = 0.001, ***p = 0.0001 in sequence, n = three independent experiments).

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