Figure 4. RelA mediates the effects of NFκB.

Neonatal cardiac fibroblasts were first transfected with either a control siRNA or a siRNA that targeted the NFκB subunit RelA. Two days later, the cells were transfected again with either the negative control miR negmiR or miR combo. The day after transfection with miRNAs, the media was replaced and the cells cultured in normal growth media for 13 days.

(A) Quantification of RelA knockdown by qPCR.

(B) Left: Cells were fixed and stained with anti-Actn2 antibodies (red). Nuclei were stained with DAPI (blue). Scale bar 100 microns. Inset pictures are at 5x magnification. Right: Quantification of immunostaining shown in B. Cells expressing Actn2 were counted and expressed as a percentage of the total cell population. N=3 independent experiments.

(C) RNA levels of the cardiomyocyte structural proteins Myh6 (αmyosin heavy chain), Actn2 (αsarcomeric actinin) and Tnni3 (cardiac troponin-I) was determined by qPCR. N=3 independent experiments.

(D) RNA levels of the endodermal marker Gata6 and the general marker of differentiation Tgfb2 were determined by qPCR. N=3 independent experiments.

Data represented as Mean ± SEM. Comparisons are made between miR combo and negative control miR (negmiR) for each group, **P<0.01, *P<0.05, ns: not significant.