Fig. 3

a–f Effects of RSPO3-KD in immortalised (im) APs. a RSPO3-KD was confirmed by qRT-PCR (Abdo, n = 7 experiments; Glut, n = 9 experiments). b RSPO3-KD results in increased doubling time in imAbdo but not imGlut APs. Doubling time of control (shCON) and RSPO3-KD (shRSPO3) imAbdo (n = 15 experiments) and imGlut APs (n = 14 experiments). c–f RSPO3-KD promotes adipogenesis more robustly in imGlut versus imAbdo cells. Representative micrographs of shCON and shRSPO3 imAbdo and imGlut cells after 14 days of differentiation are shown (c). Scale bar = 500 μm. Adipogenesis was assessed by d AdipoRed staining (four independent experiments, n = 12 replicates each, expressed as relative lipid levels), and e–f qRT-PCR of adipogenic genes (n = 5 experiments). g–j Effects of RSPO3-KD in primary (1°) APs. (g) RSPO3-KD was confirmed by qRT-PCR (n = 6 experiments). RSPO3-KD leads to increased doubling time in 1°Abdo APs (n = 6 experiments) (h) and enhanced adipogenesis in 1° Glut cells (five independent experiments) (i). Representative micrographs of AdipoRed-stained control and shRSPO3 cells at differentiation day 12 are shown (j). Intracellular lipids are stained yellow. Scale bar = 200 μm. k–m Effects of rhRSPO3 treatment on 1° AP (k) proliferation (n = 15 [cells from three female subjects]), and l–m adipogenesis under l serum-free conditions (n = 13 independent experiments [cells from seven female donors]) and m in the presence of serum (FBS) (n = 2 independent experiments, eight replicates each). veh., vehicle; R3 (5), 5 ng ml⁻¹ rhRSPO3; R3 (50), 50 ng ml⁻¹ rhRSPO3. (a, b, d–i, m) Histogram data are means ± s.e.m. k, l Error bars are median values with interquartile ranges. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical significance was assessed by two-tailed Student’s t-test (a, b, e–i, paired; d, m, unpaired, and with Bonferroni correction for m) and a two-tailed Wilcoxon signed-rank test (k, l). Source data are provided as a Source Data file.