Fig. 1: Lrig proteins regulate adipogenesis of MEFs in vitro.

Wild-type (WT) and Lrig-null (TKO) MEFs were treated with an adipogenic cocktail as described in the Methods section for the indicated times. a–c Adipogenesis of wild-type and Lrig-null MEF lines. Wild-type and Lrig-null MEFs were treated with the adipogenic cocktail for nine days followed by the quantification of adipocytes via Oil Red O staining. Shown are representative images of wild-type cells with 6% covered area (a) and Lrig-null cells with 1.7% covered area (b) (scale bar, 0.6 mm) and the quantifications of percentage area coverage for the Oil Red O stained biological replicates (n = 8 per genotype) (c). d–i Relative mRNA expression levels of Lrig1 (d), Lrig2 (e), Lrig3 (f), Cebpb (g), Pparg (h), and Ap2 (i). Cells were treated as in a-c. At the indicated time points after induction, the cells were lysed and analyzed by quantitative RT-PCR. Expression was normalized to the reference gene Rn18s. Shown in d-f are wild-type cells only, whereas both wild-type and Lrig-null cells are shown in g–i, as indicated, for eight biological replicates per genotype. j–l Lipidomic analyses. Lipids were extracted from wild-type and Lrig-null MEFs before or after eight days of treatment with the adipogenic cocktail. Lipid analysis was then performed by liquid chromatography coupled with tandem mass spectrometry. Each symbol represents one experimental replicate; shown are the results of three biological replicates per genotype with four experimental replicates each. j PCA score plots of all samples, labeled according to sample category. The variation explained by PC1 and PC2 was 39.6% and 33.6%, respectively. k The score plot of the OPLS-DA model built from the lipid profiles of the 8-day samples to determine the maximal variance between the wild-type and Lrig-null sample groups. l The corresponding loading plot explaining the contributions of different lipid species to the OPLS-DA model, indicating that triglycerides (TGs) (red triangles) in the wild-type samples were highly enriched compared to in the Lrig-null samples. The lipids are labeled according to lipid class (DG: diacylglycerol, PC: phosphatidylcholine, PE: phosphatidylethanolamine, PS: phosphatidylserine, SM: sphingomyelin, TG: triacylglycerol). m, n Role of BMP for adipogenesis in vitro. Wild-type and Lrig-null MEFs were treated as in a, without (Ctrl) or with the addition of 100 ng/ml of the BMP inhibitor noggin (Noggin) (m), or without (Ctrl) or with the addition of 50 ng/ml of BMP4 (BMP4) (n). Adipogenesis was scored through Oil Red O staining as described under a. In c–i, m and n the means of the biological replicates (c, g–i, m and n, n = 8 per genotype; d–f, n = 3) are shown by horizontal lines, and the means of the individual biological replicates analyzed by three experimental repeats are shown by dots and squares. Error bars represent the standard deviations of the means of the biological replicates. ^nsP > 0.05, *P < 0.05, **P < 0.01 (Student’s t-test).