Skip to main content

Advertisement

Fig. 4 | Cell Communication and Signaling

Fig. 4

From: miR-449a regulates insulin signalling by targeting the Notch ligand, Jag1 in skeletal muscle cells

Fig. 4

Overexpression of miR-449a and inhibition of Notch signalling promote insulin signalling. a Differentiated C2C12 cells were transfected with the miR-449a mimic (10 nM) alone or together with its inhibitor (10 nM). Control cells were transfected with the scramble (Scr). After 48 h, cells of all groups were treated with insulin (100 nM) for 20 min and on termination of incubation, they were lysed and probed for the levels of p-PI3K, PI3K (a), p-AKT, AKT (b) by western blot analysis. β-actin was used as loading control. c C2C12 cells were transfected with either the scramble or miR-449a alone or together with its inhibitor and after 48 h, were incubated in Kreb’s-Ringer bicarbonate buffer for 2 h. Cell were then pre-incubated with insulin (100 nM) for 15 min, followed by incubation with 2-NBDG (500 μM) in the presence of insulin (100 nM) for 1 h. On termination of incubation, fluorescence intensity of 2-NBDG was measured as described in ‘Materials and Methods’ section. d Differentiated C2C12 cells were incubated with the Notch inhibitor, DAPT (5,10 μM) for 24 h and the transcript levels of Notch target gene, Hes1 were quantified by qRT-PCR. Control cells were incubated in the presence of DMSO (C). 18S rRNA was used as loading control. In another experiment, cells were incubated as in (d) for 24 h and then incubated with insulin (100 nM) for 20 min and evaluated for the levels of p-PI3K, PI3K (e) and p-AKT,AKT (f). β-actin was used as the loading control. Densitometric analyses are given below the respective blots. All the experiments were done at least thrice and data presented as mean ± SEM. *p < 0.05, **p < 0.01

Back to article page