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Fig. 5 | Molecular Medicine

Fig. 5

From: H19 inhibition increases HDAC6 and regulates IRS1 levels and insulin signaling in the skeletal muscle during diabetes

Fig. 5

H19 inhibition regulates IRS1 levels in HDAC6 dependent manner and impairs insulin signaling in C2C12 cells. A Differentiated C2C12 cells were transfected with the scramble or H19 siRNA (5 nM) and treated with wither DMSO or the HDAC inhibitor, SAHA (10 µM). On termination of incubation (24 h), cells were lysed and 30 µg protein was resolved on SDS-PAGE and subjected to Western Blot analysis using anti-IRS1 antibody. HSC70 was used as the loading control. The blot presented is a representative blot and given below is the densitometric analysis of three independent experiments. B Differentiated C2C12 cells were transfected with either scramble or HDAC6 siRNA (5–25 nM) and after 48 h of transfection, cells were lysed and 1 μg of total RNA was reverse transcribed and the transcript levels of Hdac6 and Irs1 were detected by qRT-PCR. 18S rRNA was taken as the endogenous control. C Differentiated C2C12 cells were transfected as in “B” and after 48 h of transfection, 20 µg of protein was resolved on SDS-PAGE and probed using anti-HDAC6 and anti-IRS1 antibodies. HSC70 was used as the loading control. The blot presented is a representative figure and given below is the densitometric analysis of three independent experiments. D Differentiated C2C12 cells were transfected with the scramble or H19 siRNA (5 nM) alone or together with HDAC6 siRNA (25 nM). After 48 h of transfection, cells were lysed and 1 μg of total RNA was reverse transcribed and the transcript levels of Irs1 were determined by qRT-PCR. 18S rRNA was taken as the endogenous control. Also, after a similar transfection, 20 µg of protein from scramble or H19 siRNA alone or together with HDAC6 siRNA transfected cells was resolved on SDS-PAGE and probed using anti-IRS1 antibody. HSC70 was used as the loading control. The blot presented is a representative figure and given below is the densitometric analysis of at least five independent experiments. C2C12 cells were transfected with either control vector or HDAC6 cloned vector (0.5 µg and 1 μg) and 48 h post-transfection, cells were lysed and the transcript levels of Hdac6 (E) or Irs1 (F) were determined by qRT-PCR. 18S rRNA was used as the endogenous control. G Differentiated C2C12 cells were transfected with either control vector or HDAC6 overexpression vector (0.5 µg and 1 μg) and 48 h post-transfection, cells were lysed and 20 µg lysate was resolved on SDS-PAGE and probed using anti-HDAC6 and anti-IRS1 antibody. HSC70 was used as the loading control. The blot presented is a representative figure and given below is the densitometric analysis of four independent biological replicates. Differentiated C2C12 cells were transfected with either the scramble or H19 siRNAs and after 48 h of transfection, cells were treated with insulin (100 nM, 20 min). On termination of incubation, cells were lysed and probed for IRS1, pIRS1 (H) and Akt, pAkt (I) protein levels by Western Blot analysis using specific antibodies. Vinculin was taken as the loading control. All experiments were done at least thrice and densitometric analyses of the same are provided. Values are means ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

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