Fig. 4

LncRNA H19 inhibition decreases IRS1 levels in C2C12 cells. A Putative acetylation marks as identified on the regulatory promoter regions of PRKAB2, PFKFB3, SREBF1, SOCS2, IRS1 and PPP2R5B using the UCSC genome database. Regions enclosed within the red box upstream to each gene represent the site with potential histone acetylation (K9 and K27) marks, red arrows represent the orientation of the gene. B Total RNA was isolated from skeletal muscle tissues of normal db/ + and diabetic db/db mice (n = 4) and 1 µg RNA was reverse transcribed and transcript levels of Prkab2, Pfkfb3, Srebf1, Socs2, Irs1 and Ppp2r5b were evaluated by qRT-PCR. 18S rRNA was taken as the normalization control. C C2C12 cells were differentiated and transfected with either the scramble or H19 siRNA (5 nM) and after 48 h, total RNA was isolated, reverse transcribed and the transcript levels of Prkab2, Pfkfb3, Srebf1, Socs2, Irs1 and Ppp2r5b were assessed using gene specific primers. 18S rRNA was taken as the endogenous control. Experiments were done in at least four independent sets. D Cells transfected as in “C” were lysed and 20–30 µg protein was run on SDS-PAGE and subjected to Western Blot analysis using anti-IRS1 antibody. HSC70 was taken as the loading control. Experiments were done in four independent sets. E Skeletal muscle tissue lysates (30 µg) of normal db/ + and diabetic db/db mice (n = 6) were subjected to Western Blot analysis using anti-IRS1 antibody. HSC70 was taken as the loading control. Given below is the densitometric analysis of the normalized values of six mice in each group. Values presented are means ± SD of four independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001¸ns: non-significant