Fig. 5

Tip60-mediated lipin 1 acetylation leads to lipin 1 translocation from cytosol to ER membranes. a Attenuated translocation of lipin 1 from cytosol to ER membranes after Tip60 depletion. Representative images (n = 3 replicate experiments) of 3T3-L1 adipocytes expressing control shRNA (ctrl) or shRNA targeting Tip60 (Tip60 KD) treated with BSA or OA for 2 h. Calnexin is an ER/microsomal marker. Mander’s overlap coefficients between lipin 1 and Calnexin were graphed (n = 30 for each group). Scale bar, 10 μm. b Depletion of Tip60 impairs lipin 1 ER localization. Western blotting of proteins in subcellular fractions of 3T3-L1 adipocytes expressing control shRNA (ctrl) or shRNAs targeting Tip60 treated with or without OA for 3 h. Calnexin, microsomal (Mic) marker; β-tubulin, cytosol (Cyt) marker. c Impairment of lipin 1 ER localization in adipocytes derived from Tip60^SA/SA MEFs. Adipocytes (differentiation day 8) derived from WT or Tip60^SA/SA MEFs were treated with OA for 1–3 h and analyzed by fractionation, followed by western blotting. d Depletion of Sirt1 promotes lipin 1 ER localization. e Knockdown of Lpin1 in 3T3-L1 adipocytes. Western blotting of proteins of 3T3-L1 adipocytes expressing control shRNA (ctrl) or shRNA targeting Lpin1. f, g Acetylation-dependent ER localization of lipin 1 and DAG/TAG synthesis. Western blotting of proteins (f) or DAG, TAG and PC synthesis rates (g) of 3T3-L1 adipocytes expressing shRNA targeting Lpin1 reconstituted with vector, WT-lipin 1 or 2KR-lipin 1 after OA treatment (n = 4 experiments). Error bars denote SEM. Statistical analysis was performed by ANOVA followed by Tukey in a, g. **P < 0.01; N.S. not significant. Uncropped blots can be found in Supplementary Fig. 6