Fig. 6: LXN negatively regulates PPARɤ ubiquitination by inhibiting FABP4 expression. | Cell Death & Disease

Fig. 6: LXN negatively regulates PPARɤ ubiquitination by inhibiting FABP4 expression.

From: Latexin deficiency attenuates adipocyte differentiation and protects mice against obesity and metabolic disorders induced by high-fat diet

Fig. 6

A, B Primary preadipocytes isolated from WT and LXN−/− mice were cultured with normal medium (NM) or differentiation medium (DM) for three days. Cell extracts were subjected to Western blot analysis with the indicated antibodies (A), and the relative protein levels of LXN, PPARɤ and FABP4 were assessed (B). C, D 3T3-L1 cells were transfected with scramble siRNA or LXN siRNA. Twelve hours later, the medium was changed, and the cells were cultured in the differentiation medium containing 20 µmol/L BMS (FABP4 inhibitor) for 3 days. The cell extracts were then subjected to Western blot analysis with the indicated antibodies (C), and the relative protein levels of LXN, PPARɤ and FABP4 were assessed (D). E-G Normal medium-cultured 3T3-L1 cells were transfected with 0, 1, 3, 5 µg Flag-LXN plasmid, respectively, for 48 h. The cell extracts were then subjected to Western blot analysis with the indicated antibodies (E), and relative quantification of FABP4 and PPARɤ were presented (F). The relative mRNA levels of FABP4 and PPARɤ were determined by qPCR (G). H qPCR determine the relative mRNA level of FABP4 (left) and PPAR ɤ (right) in WT and LXN-/- primary preadipocytes under normal medium or differentiation medium condition. I WT and LXN-/- primary preadipocytes were treated with CHX (350 mmol/L) and harvested at the indicated times after CHX addition. Immunoblots of cell extracts with antibodies directed against PPARɤ, LXN, and β-actin are shown (left). Half-life analysis of PPARɤ protein, relative to time 0 (right). J Overexpression of LXN by lentivirus in LXN-/- primary preadipocytes followed by CHX treatment for the indicated times. Immunoblots of cell extracts with antibodies directed against PPARɤ, LXN, and β-actin are shown (left). Half-life analysis of PPARɤ protein, relative to time 0 (right). K 3T3-L1 cells were transfected with scramble siRNA or LXN siRNA for 48 h. After that the cells were treated with 20 µmol/L BMS for 12 h, and then the cells were cultured with differentiation medium. After 48 h, the cells were treated with MG132 (0.1 µmol/L) for another 12 h. Cell lysates were immunoprecipitated with an anti-ubiquitin or anti-PPARɤ antibody, and the immunoprecipitates were immunoblotted with antibodies directed against ubiquitin or PPARɤ. All results were presented as mean ± SD. *P < 0.05, and **P < 0.01, *** P < 0.001, ns: no significance.

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