Fig. 4

a Light microscopy of abdominal and gluteal DFAT stable cell lines after 14 days adipogenic differentiation. Scale bar = 100 μm. b, c Expression of RSPO3 in DFAT stable cell lines at day 15 of adipogenic differentiation following ~ 48-h treatment with 0.05 µg ml⁻¹ doxycycline versus vehicle in hormone-free basal media (n = 5 experiments). d, e Insulin-stimulated glucose uptake in DFAT cells relative to basal glucose uptake following ~ 48-h treatment with 0.05 µg ml⁻¹ doxycycline or vehicle in hormone-free basal media (n = 12, from four independent experiments). f, g Isoproterenol-stimulated glycerol release in DFAT cells relative to basal glycerol release following ~ 48-h treatment with 0.05 μg ml⁻¹ doxycycline or vehicle (n = 12, from four independent experiments). b–g Histogram data are means ± s.e.m. *p < 0.05, **p < 0.01. Statistical significance was assessed by two-tailed paired (b, c) and unpaired (d–g) Student’s t-test comparing doxycycline versus vehicle treatment. h, i Transcriptional profiling of in vitro differentiated abdominal and gluteal DFAT cells following 48-h doxycycline-induced RSPO3-KD. h Number of differentially expressed (DE) genes. FDR p < 0.05. i Gene-set enrichment analysis results of DE genes in gluteal DFAT dox-induced RSPO3-KD cells showing the top 10 GO biological processes. j, k Effect of doxycycline-induced RSPO3-KD in differentiated j abdominal, and k gluteal cells on apoptosis in the presence of indicated concentrations of rhTNFα (ng ml⁻¹). Apoptosis was assayed using Caspase Glo 3/7 reagent. Results are shown as a % of luminescence of non-dox (vehicle)-treated cells in the presence of the same concentration of rhTNFα (n = 7 independent experiments). Statistical significance was assessed by a two-tailed Wilcoxon signed-rank test comparing dox and non-dox treated cells. Solid symbols, tet-shCON cells; open symbols, tet-shRSPO3 cells; circles, no rhTNFα; squares, 10 ng ml⁻¹ rhTNFα; triangles, 100 ng ml⁻¹ rhTNFα. Error bars are median values with interquartile ranges. Source data are provided as a Source Data file.