Extended Data Fig. 1: Lpcat3 is an evolutionarily conserved PPARγ target gene.
From: Dietary control of peripheral adipose storage capacity through membrane lipid remodelling
(a) qPCR analysis of Lpcat1-4 mRNA levels in eWAT and iWAT of 12-week-old NCD-fed wild-type mice (n = 5, 5), and (b) in iWAT from wild-type mice fed low-fat diet (LFD) with or without rosiglitazone (Rosi; 50 mg kg–1) for 14 days (n = 7, 7). (c) Immunoblot analysis of LPCAT3 protein levels in iWAT lysates from (b). Tubulin served as a sample processing control. (d) Immunoblot analysis of LPCAT3 protein levels and known adipogenic markers during the course of 10T1/2 adipocyte differentiation. Calnexin served as a loading control for LPCAT3. (e) Sequence alignment of conserved PPAR and LXR response elements in the Lpcat3 promoter across multiple species. The PAM sequence of sgRNAs targeting Lpcat3-PPRE and LXRE are underlined in red. (f) ChIP-qPCR analysis of PPARγ (or normal rabbit IgG) occupancy at Lpcat3, Plin1, Fabp4, Agpat2, or Aqp7 PPRE elements in wild-type and ΔPPRE 10T1/2 adipocytes (n = 3/group; 100–mm plate per replicate from 3 independent experiments). A region of the 36b4 promoter served as a negative control. (g) PCR analysis of a 700-bp genomic segment flanking the PPRE/LXRE sites in the Lpcat3 promoter region of WT, ΔPPRE, and ΔLXRE clonally-derived cell lines, revealing no major deletions introduced by CRISPR/Cas9 gene editing. Primer anneal sites in the Lpcat3 promoter are shown in orange, indels in green (insertions) or red (deletions). (h) Immunoblot analysis of LPCAT3 protein levels in WT, ΔPPRE, and ΔLXRE 10T1/2 adipocytes differentiated for 4 days. Calnexin served as a loading control for LPCAT3. Data are presented as mean ± SEM. ***P < 0.001 by Welch’s t-tests with FDR correction using the Benjamini, Krieger, and Yekutieli procedure (b); or one-way ANOVA with Tukey’s multiple comparisons test (f).
