Fig. 4. DS0908 and DS0950 culture supernatants activate thermogenesis via PKA-p38 MAPK signaling in C3H10T1/2 MSCs.
(A) Phosphorylated protein expression levels of PKA substrates, p-p38 MAPK, p-CREB and p-AMPK after incubation with DS0908 and DS0950 for 10, 30, and 60 min. The confluent C3H10T1/2 MSCs were serum-depleted for 8 h, incubated with DS0908 and DS0950 for the indicated periods, then the phosphorylated proteins were detected. (B) Phosphorylation levels of PKA and p38 MAPK after treatment with 8-br-cAMP (PKA activator), H89 (pan-PKA inhibitor) and SB 203580 (p38 MAPK inhibitor) with or without DS0908 and DS0950. (C) mRNA expression levels of p38 MAPKα in Pkaα and p38 MAPKα-knockdown cells, and their downstream thermogenic genes Ucp1, Pgc1α, Prdm16 and Pparγ after silencing of Pkaα and p38 MAPKα and treatment with DS0908 and DS0950 (siPkaα/sip38 MAPKα + DS0908 or DS0950 group) and control siRNA and treatment with DS0908 or DS0950 (siCont + DS0908 or DS0950 group). (D) Protein expression levels (UCP1, PGC1α, PRDM16 and PPARγ) were measured after silencing of Pkaα and p38 MAPKα and treatment with DS0908 or DS0950 (siPkaα/sip38 MAPKα + DS0908 or DS0950 group) and control siRNA and treatment with DS0950 or DS0908 (siCont + DS0908 or DS0950 group). The gene knockdown experiments were designed as siCont vs. siCont + DS0908 or DS0950, siPkaα vs. siPkaα + DS0908 or DS0950 and sip38 MAPKα vs. sip38 MAPKα + DS0908 or DS0950. Post silencing with the siRNA, C3H10T1/2 mesenchymal stem cells (MSCs) were differentiated as described in Methods. Tbp was used as an internal control gene and β‐actin as a protein loading control. The data from three individual experiments are expressed as the average ± standard error mean (SEM). *, **, *** and ns indicate p < 0.05, < 0.01, < 0.001 and non-significant, respectively, to express the statistically significant differences between the control (MDI) and the treatment groups in the figures. The protein band intensities were measured using ImageJ. Adipogenic differentiation medium, MDI: 0.5 mM IBMX, 1 μM dexamethasone and 10 μg/ml insulin; 1 μM Rosiglitazone (Rosi); DS0908 = B. bifidum DS0908; DS0950 = B. longum DS0950.