Fig. 3: Identification of Rspo2 as a new marker of P3.
From: Identification of a regulatory pathway inhibiting adipogenesis via RSPO2
a, Cd142 Vap1 and Rspo2 expression in eP3-depleted SVF, VAP1+CD142+ APCs and eP3. Data show the mean ± s.e.m., n = 6 biological replicates. Cd142: F(2,15) = 88.9; Vap1: F(2,15) = 686.4; Rspo2: F(2,15) = 79.5 using one-way ANOVA. b–d, The ratio (b) and representative images (d) of adipocytes after knocking down of Rspo2 in ingWAT SVF. Rspo2 mRNA expression (c) 48 h after transfection. Data show mean ± s.e.m., analyzed by two-tailed Student’s t-test. n = 2 biological replicates (b), n = 4 biological replicates (c). Ctrl, control. e–h, Scheme of Transwell co-culture experiments (e). The ratio (f) and representative images (h) of CD142− cells on differentiation day 8. Rspo2 mRNA levels (g) in siRNA-transfected eP3. Data show mean ± s.e.m., analyzed with two-tailed Student’s t-test, n = 2 biological replicates (f,g). i–k, Experimental scheme (i) for rec.RSPO2 treatment experiment. The ratio (j) and microscopy images (k) of adipocytes in SVF-treated ± rec.SPO2. Data shown as mean ± s.e.m., n = 6 independent wells. Data were analyzed with one-way ANOVA followed by Tukey’s multiple comparisons test. F(6,35) = 10.18. Spearman r correlation between RSPO2 level in medium and adipocyte ratio (j right). NS, not significant. l–n, Experimental scheme (l) for knocking down of Lgr4, Lgr5 and Lgr6 in ingWAT SVF treated with or without rec.RSPO2. Representative images (m) and the ratio (n) of mature adipocytes per well. Data shown as mean ± s.e.m., n = 6 independent wells. F(3,30) = 1.07, P = 0.377 using two-way ANOVA. Multicomparsion between groups was performed by two-stage step-up method with FDR = 0.05. o, Heat map of Lgr4, Lgr5 and Lgr6 expression in eP1 and eP2 cells, n = 3–5 biological replicates. p–q, Ratio (p) and representative images (q) of adipocytes in cells treated ± rec.RSPO2. Data shown as mean ± s.e.m., n = 6 independent wells. F(2,20) = 26.22, P < 0.0001 using two-way ANOVA. Multicomparison between groups was performed by two-stage step-up method with FDR = 0.05. In all panels, nuclei were stained with Hoechst 33342 (blue) and lipids were stained with LD540 (yellow). Scale bars, 100 μm.
