Figure 5: MP^Hh+ and GSA-10 induce inhibition of adipocyte differentiation through activation of Ampk signalling pathway.

(a) Western-blot showing increased phosphorylation of Ampk in MP^Hh+ (10 μg/mL) and GSA-10-treated 3T3-L1 preadipocyte cells for 24 h. Following compound concentrations were used: SAG (200 nM), GSA-10 (10 μM), MP^Hh+ (10 μg/mL) and recShh (0.5 μg/mL). β-actin immunoblot serves as a loading control. Unprocessed immunoblots, from which the presented images were cropped are presented Fig. S8. Immunoblots quantification was performed from at least n = 3 independent experiments. P-Ampk/total Ampk ratio are presented as experimental data dots with median plus range from n = 3–7 independent experiments. (b) Lkb1 silencing prevents from MP^Hh+ and GSA-10-induced anti-adipogenic effects as illustrated by restored lipid accumulation. 3T3-L1 stably expressing scrambled shRNA (Scr), Camkk2 shRNA (Camkk2 Kd) or Lkb1 shRNA (Lkb1 Kd) and the control cell line were treated with 10 μg/mL MP^Hh+ or 10 μM GSA-10 during the induction cocktail in minimal induction medium. Representative ORO-stained images of 3T3-L1-treated cells (day 6 of differentiation) are presented, n = 2 independent experiments. (c,d) Lkb1 knockdown abrogates inhibition of differentiation induced by MP^Hh+ and GSA-10, whereas Camkk2 knockdown has no effects. Camkk2 Kd or Lkb1 shRNA 3T3-L1 cells were treated or not treated (Ctrl) with 10 μg/mL MP^Hh+ or 10 μM GSA-10 during the induction period. PPARγ and aP2 mRNA expression was analysed by real-time qPCR analysis. Lkb1 Kd abrogates inhibition of differentiation induced by MP^Hh+ or GSA-10 as illustrated by reversed mRNA expression of PPARγ and aP2. (c) whereas inhibition of the mRNA expression of these two genes is still maintained in Camkk2 Kd cells. (d). Conversely, neither Lkb1 nor Camkk2 silencing reverse anti-adipogenic effects of SAG (200 nM) or recShh (0.5 μg/μl) illustrated by conserved inhibition of adipocyte markers (c,d).