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. 2016 Mar 24;6:23479. doi: 10.1038/srep23479

Figure 3. Adipogenesis inhibition induced by MPHh+ and by the Smo agonist GSA-10 is independent of the Hh canonical pathway.

Figure 3

(a) Anti-adipogenic effects of MPHh+ or GSA-10 are not dependent on the presence of IBMX or dexamethasone in the induction medium, contrary to that of SAG, purmorphamine or recShh. ORO staining of 3T3-L1 cells induced with classical (IBMX/Dex/Ins) or minimal (Rosi/Ins) differentiation cocktails in the absence (Ctrl) or presence of SAG (200 nM), purmorphamine (purmo, 10 μM) and recShh (0.5 μg/mL), 10 μg/mL MPHh+ or MPHh− and GSA-10 (10 μM). Representative images of ORO-stained cells are shown (scale bar: 50 μm). (b) Differential effects of MP and Smo agonists SAG and GSA-10 on Gli-dependent luciferase reporter activity in Shh-light2 cells was measured in the presence of SAG (1 μM), GSA-10 (3 μM), MPHh+ (10 μg/mL) and MPHh− (10 μg/mL). SAG but not MPHh+, MPHh− nor GSA-10 induces Gli-luciferase activity, expressed as fold change (FC) compared to cells treated with vehicle alone.(c) mRNA expression levels of Hh pathway genes tested by real-time qPCR in response to MPHh+, MPHh− and Smo agonists SAG and GSA-10 in minimal induction medium. Compound concentrations were similar to those in (a). SAG and recShh induced a strong induction of Gli1 and Ptch mRNA expression (10–40 fold increase relative to control cells, log scale) whereas MPHh+ or GSA-10 display no inductive effect on Gli-transcriptional activity. (d) Smo knockdown abrogates inhibition of differentiation induced by SAG, MPHh+ and GSA-10. 3T3-L1 cells stably expressing scrambled shRNA (Scr) or Smo shRNA (Smo Kd) were treated in minimal induction medium with SAG (200 nM), MPHh+ (10 μg/mL) or GSA-10 (10 μM) during the induction period. ORO staining and quantification show reversion of adipocyte differentiation blockade induced by all Hh signalling inducers in Smo Kd 3T3-L1 compared to Scr 3T3-L1 cells. (e) 5E1 Ab blocks anti-adipogenic effects of recShh, but not those induced by MPHh+. 3T3-L1 cells were incubated with recShh (0.5 μg/mL) or MPHh+ (10 μg/mL), both reagents preincubated with or without 5E1 Ab (10 μg/mL) for 30 min. Representative images are shown, n = 2 independent experiments (scale bar: 100 μm).