Fig. 2

H19 inhibition regulates HDAC6 levels in C2C12 cells. A Differentiated C2C12 cells were transfected with either the scramble or H19 siRNA (1–5 nM) and after 48 h, total RNA was isolated and the transcript levels of H19 were assessed by qRT-PCR. 18S rRNA was used as the loading control. B Differentiated C2C12 cells were transfected with either the scramble or H19 siRNA as in “A” and after 48 h, cells were lysed and 90 µg cell lysates were used to assay HDAC activity as described in the “Material and methods” section. HDAC activity is expressed as µM of substrate deacetylated per µg of protein. C Total RNA was isolated from the C2C12 cells transfected as in “A” and the transcript levels of HDAC 1–6 were evaluated by qRT-PCR. 18S rRNA was used as the loading control. C2C12 cells transfected as in “A” were lysed and lysates (20–30 µg) were subjected to Western Blot analyses to evaluate the levels of HDAC 1–6. HSC70 was used as the loading control (D–I). J 20 µg of lysate from scramble and H19 siRNA transfected C2C12 cells was subjected to western blot analysis to probe the expression of pHDAC6-Ser22. HSC70 was used as the loading control. Figures presented are representative images and values are means ± SD of at least three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns: non-significant