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. 2017 Dec 11;8(12):3201. doi: 10.1038/s41419-017-0070-z

Fig. 3. CACUL1 represses PPARγ-mediated transcriptional activity and adipogenesis.

Fig. 3

a Endogenous interactions among PPARγ, SIRT1, and CACUL1 in 3T3-L1 preadipocytes. IP was performed using an anti-PPARγ antibody and bound protein complex was visualized with WB using an anti-SIRT1 or anti-CACUL1 antibody. b Effect of CACUL1 overexpression on PPARγ transcriptional activity. HEK293 cells were transiently transfected with a PPRE-Luc reporter and increasing amounts of FLAG-CACUL1 with Myc-PPARγ in the presence (or absence) of 5 µm rosiglitazone. Cell extracts were subjected to luciferase assays. Data represent means±standard deviation (SD; n = 3) from three independent experiments (**P < 0.01). c Effect of CACUL1 depletion on PPARγ activity. Luciferase assays were performed as described using extracts of HEK293 transfected with sh-CACUL1. Data represent means±SD from three independent experiments (*P < 0.05, **P < 0.01). d Effect of CACUL1 overexpression on adipogenesis. 3T3-L1 cells were transfected with FLAG-CACUL1 (or FLAG) and selected against G418 for stable expression of CACUL1. Selected cells were subjected to differentiation for 8 days. Lipid accumulation was visualized by Oil Red O staining and light microscopy at ×100. The amount of lipid was quantified using a spectrophotometer at 500 nm. Data represent means±SD from three independent experiments (**P < 0.01). e Effect of CACUL1 knockdown on adipogenesis. 3T3-L1 cells were transfected with sh-CACUL1 (or sh-Luciferase) and selected against Hygromycin B. (**P < 0.01). f, g Effects of CACUL1 on the expression of PPARγ target genes. Stable 3T3-L1 cells with either CACUL1 overexpression (f) or knockdown (g) were subjected to differentiation for 6−8 days. Subsequent RT-qPCR was performed using primer sets for two PPARγ target genes associated with adipogenesis, aP2 (also called Fabp4) and LPL. Fold mRNA expression was normalized to GAPDH expression (*P < 0.05, **P < 0.01)