Fig. 5: mTOR/RXR/PPARɤ signaling pathway is required for the upregulation of LXN during adipogenesis.

A, B 3T3-L1 cells were cultured in differentiation medium for 0, 3, 5, 7 d. Cell extracts were subjected to Western blot analysis with the indicated antibodies (A) and then quantified and normalized (B). *P < 0.05, and **P < 0.01 vs. 0 d. C, D 3T3-L1 cells were pre-treated with 10µmol/L perifosine (AKT inhibitor) or 20nmol/L rapamycin (mTOR inhibitor) for 12 h. After that, the cells were cultured with normal medium or differentiation medium for 3 d. The cell extracts were then subjected to Western blot analysis with the indicated antibodies (C), and the relative protein levels of PPARɤ and LXN were assessed (D). E, F 3T3-L1 cells were pre-treated with 10 µmol/L GW9662 (PPARɤ antagonist) for 2 h, and then cells were cultured with normal medium or differentiation medium for 3 d. The cell extracts were then subjected to Western blot analysis with the indicated antibodies (E), and the relative protein levels of PPARɤ and LXN were assessed (F). G, H Normal medium or differentiation medium-cultured 3T3-L1 cells were treated with 20 µmol/L 3BDO (mTOR agonist) for 12 h followed by treatment with 20 µmol/L PA452 (RXR antagonist) for an additional 60 h. to assess PPARɤ and LXN protein levels. The cell extracts were then subjected to Western blot analysis with the indicated antibodies (G), and the relative protein levels of PPARɤ and LXN were assessed (H). I 3T3-L1 cells were cultured in differentiation medium with GW9662, rapamycin or PA452, respectively, for 3 d. Cultured cells were subjected to Oil Red O staining. Scale bar = 50 μm. J Prediction of PPARɤ and PPARɤ::Rxra binding sites in LXN promoter region (~3000 bp) by JASPAR CORE database (https://jaspar.genereg.net/). Pink, represents the predicted PPARɤ binding region; Green, represents the predicted PPARɤ::Rxra binding region; K 3T3-L1 cells cultured in normal medium (NM) and differentiation medium (DM) with or without GW9662 (PPARɤ antagonists) treatment for 3 d, and the binding activity of PPARɤ to LXN promoter in 3T3-L1 cells was determined by ChIP assay. L Schematic diagram to demonstrate the mechanism by which LXN was upregulated during preadipocyte differentiation. All results were presented as mean ± SD. *P < 0.05, and **P < 0.01, ns: no significance.