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. 2019 Dec 27;9:20152. doi: 10.1038/s41598-019-56817-w

Figure 4.

Figure 4

LKU4 facilitates the interaction between PPARγ and PGC-1α in iWAT and 3T3-L1 adipocytes, which stimulates Ucp1 expression. (A) IP analysis of the interaction between PPARγ, PGC-1α, and RIP140 in iWATs of HFD- and HFD-LKU4-fed mice using the indicated antibodies (Cropped blots were used). (B) Day 8 3T3-L1 adipocytes cotransfected with PPARγ, PGC-1α, and RIP140 expression plasmids were treated with LKU4-CM or rosiglitazone (5.5 μM) for 24 h, following which IP analysis was performed with the indicated antibodies (Cropped blots were used). (C) A ChIP assay was performed on day 7 3T3-L1 adipocytes cotransfected with PPARγ, PGC-1α, and RIP140 expression plasmids in the presence or absence of LKU4-CM and rosiglitazone (5.5 μM). Protein/DNA complexes were immunoprecipitated with the indicated antibodies and the Ucp1 promoter region containing a PPRE (−2551 to −2541 bp) was PCR-amplified from immunoprecipitated DNA. Con vs LKU4-CM; *P < 0.05 (D) Luciferase activity in HEK293T cells cotransfected with a reporter plasmid (pGL3-Ucp1-Luc) and the indicated expression plasmids and/or siRNA. After 12 h of transfection, cells were treated either with control medium or LKU4-CM for 48 h. (E) Day 6 3T3-L1 adipocytes were transfected with the indicated expression plasmids and/or siRNA. After 12 h of transfection, cells were incubated in the presence or absence of LKU4-CM for 48 h and harvested for RT-qPCR analysis. *P < 0.05, **P < 0.005.