(a), Subcutaneous white pre-adipocytes (left) and C2C12 myoblasts (right) were infected by retrovirus expressing Prdm16 two days before differentiation. 5 days after differentiation, real-time PCR were performed to examine the expression of miR-193b, miR-365, and as a control miR-223. n=3. (b), Primary brown preadipocytes were infected by retrovirus expressing shRNA targeting Prdm16 two days before differentiation. By D5 of differentiation, real-time PCR was used to examine the mRNA level of Prdm16 (left), and miR-193b, miR-365, and the control miR-223 (right). n=3. (c), Real-time PCR analysis for Pparα in primary brown adipocytes retrovirally expressing shRNA for Prdm16. (d), Real-time PCR analysis of miR-193b, miR-365, Prdm16 and Ppara expression levels during adipogenesis of primary brown adipocyte cultures. n=3. (e), ChIP analysis for the interaction between Ppara and the promoter region of miR-193b-365. Immortalized brown adipocyte cultures (Day 5) were fixed and sheared by ultrasonication. Immunoprecipitation was performed with anti-Pparα and control IgG. Recovered DNA was amplified by 3 pairs (P1, P2 and P3) of primers designed to span the 1Kb promoter region. Input samples are in the top panel and immunoprecipitated ones in the bottom. (f), Primary brown preadipocytes were transfected with siRNA targeting Pparα, and differentiated for 3 days. RT- PCR was performed to examine the expression of Pparα, and miR-193b and miR-365. Pparγand miR-223 were used as controls. n=3. (g), RT-PCR was performed to examine the expression of miRNAs in brown adipose tissue isolated from Pparα knockout mice (8 week old, Male). Age-matched wild- type mice were used as control. n=6. * P < 0.05, Student’s t-test; Means ± SEM.