Fig 1: Rspo2 inhibits transition of eP1 cells to eP2 cells.a–f, Experimental scheme (a) for transplantation of tdTomato+ eP1 cells into inguinal adipose tissue of wild-type (WT) mice. FACS analysis (b) of VAP1 and CD142 expression in tdTomato+ eP1 cells 10 d after transplantation. Expression of P1 marker genes (c) (Cd55, Dpp4, Pi16 and Psck6), P2 marker genes (d) (Vap1, Icam1, Col4a1 and Sparcl1), Pparg and Cebpa (e) and P3 marker genes (f) (Cd142, Gdf10, Clec11a and Igfbp3) in eP1 cells (from donor mice), eP2 cells (from donor mice), eP3 cells (from donor mice), VAP1+ cells (derived from implanted eP1 cells) and VAP1- cells (derived from implanted eP1 cells). Data are shown as mean ± s.e.m., n = 4 biological replicates. g–m, Experimental scheme (g) for injection of AAVs into ingWAT for overexpression of RSPO2. Western blot images (h) and quantification (i) of RSPO2 protein and Rspo2 mRNA (j) in ingWAT. FACS analysis of eP1/SVF (k), eP2/SVF (l), CD55+VAP1+ (m) in ingWAT. Data are shown as mean ± s.e.m., n = 6 mice (h,i), n = 5–6 mice (j), n = 5 mice (k–m). Data were analyzed using two-tailed Student’s t-test. n,o, Experimental scheme (n) for transplantation of tdTomato+ eP1 cells into RSPO2 overexpression mice. FACS analysis of (VAP1+:tdTomato+) cells in tdTomato+ eP1 cells (o). Data are shown as mean ± s.e.m., n = 5 biological replicates. Data were analyzed using a two-tailed paired Student’s t-test.Source data
Fig 2: Rspo2 inhibits adipogenesis of eP1 cells in vivo.a–f, Experimental scheme (a) for cell transplantation in Matrigel. Rspo2 expression in eP1 Matrigel plugs and in eP2 Matrigel plugs (b). Quantification of adipocytes and cell number in eP1 Matrigel plugs (c) and eP2 Matrigel plugs (e). Representative hematoxylin and eosin (H&E) staining of eP1 Matrigel plugs (d) and eP2 Matrigel plugs (f). Data show mean ± s.e.m., n = 3 biological replicates (b), n = 5 biological replicates (c,e). Data analysis was performed using a two-tailed Student’s t-test. Scale bar, 100 µm. g–k, Experimental scheme for overexpression of RSPO2 in AdipoCre-NucRed mice fed with HFD or chow diet. Western blot images (h) and quantification (i) of RSPO2 protein in liver and ingWAT; HSP90 bands were used as loading control. Quantification of adipocyte numbers in ingWAT (j) and visWAT (k) of mice shown in g. Data are shown as mean ± s.d., n = 6 mice. Data analysis was performed by two-tailed Student’s t-test (i) and one-way ANOVA (j,k). In j, Total cell number, F(3,20) = 14.4, P < 0.0001; adipocyte, F(3,20) = 15.50, P < 0.0001; non-adipocyte, F(3,20) = 14.1, P < 0.0001. In k, total cell number, F(3,20) = 14.4, P < 0.0001; adipocyte, F(3,20) = 15.50, P < 0.0001; non-adipocyte, F(3,20) = 14.1, P < 0.0001. l–o, Experimental scheme (l) for overexpression of RSPO2 in ingWAT by injection of AAV into ingWAT of AdipoCre-NucRed mice. Western blot images (m) and quantification (n) of RSPO2 protein in ingWAT of mice shown in l. HSP90 bands were used as loading control. Quantification of cell numbers by quantitative PCR in ingWAT (o). Data shows mean ± s.d., n = 5–6 mice. Data were analyzed using a two-tailed Student’s t-test.Source data
Fig 3: Selection and identification of Aregs’ effectors.a–c.) Scheme of identification of Aregs marker genes candidates (a), and their expression in eP1 and eP2 cells bulk RNAseq data, n = 3-5 biological replicates (b). mRNA expression in eP3 and eP3-depleted SVF (c). n = 6 biological replicates, data show the mean ± SEM., and analyzed by two-tailed Student’s t-test. d) Adipocyte ratio in ingWAT SVF after knocking down eP3 marker genes by siRNA. n = 3 biological replicates; data shown as mean ± SEM. and analyzed by two-tailed Student’s t-test (compared to Ctrl group). e) Adipocyte ratio in CD142- cells co-cultured with eP3. n = 3 biological replicates; data shown as mean ± SEM. and analyzed by two-tailed Student’s t-test (compared to Ctrl group). f) Comparison of adipogenesis of CD142- cells after knocking down of Spink2, Rspo2, Cgref1 and Serpinb6c in eP3 cells in transwell co-culture experiments. Adipocyte ratio normalized to ctrl group. n = 3 biological replicates; data is presented as mean ± SEM and analyzed by one way-ANOVA test. F(3,20)=2.025, P = 0.143. g) Adipocyte ratio in eP3 after knocking down Rspo2 by siRNA, n = 3 biological replicates. Representative images of adipocytes on differentiation day 7. h) RSPO2 conc. in cell culture medium, n = 4 biological replicates. Data is presented as mean + /- SEM and was analyzed by one way-ANOVA test. F(3,12)=75; P < 0.0001. i) Quantification of cell number in Fig. 3j. n = 6 independent wells; data show the mean ± SEM, analyzed by one way-ANOVA test. j) Feature plots of Lgr4 in 10xscRNAseq of ingWAT Lin- cells9. k) Pathway enriched in eP1 cells by Enrichr analysis. P-value is computed using the Fisher exact test. l) Heatmap of log2 fold changes of Wnt signaling related genes in eP1 and eP2 cells. Each row represents 1 gene; each column represents one replicate. m) Pathway enriched in eP2 cells by Enrichr analysis. P-value was computed using the Fisher exact test. n) Heatmap of log2 fold changes of adipogenesis related genes in eP1 and eP2 cells. Each row represents 1 gene; each column represents one replicate. o) Quantification of cell number per field in Fig. 3n. Data show the mean ± SEM, n = 6 independent wells. p) Lgr4-6 mRNA level in cells in Fig. 3n. Data shown as mean ± SEM, n = 6 independent wells. Statistical analysis was performed by two-tailed Student’s t-test. q) Quantification of cell numbers per field in Fig. 3p. Data shown as mean ± SEM, n = 6 independent wells. r) Experimental scheme of treatment cells with rec.RSPO2 during adipogenesis day3 to day6. s) Quantification of adipocytes per well (left) and cell number (right) ± rec.RSPO2 during day3 to day6. Data shown as mean ± SEM, n = 6 independent wells. t–w) SVF cells treated with 0.5ug/ml rec.RSPO2 for 0-24 h. Western blot images (t) and quantification (u) of beta-Catenin and beta-Actin in SVF. Data shown as mean ± SEM, n = 3 biological replicates. F(3,8)=3.85, P = 0.057 by one way ANOVA. Multiple comparison between groups was performed using Tukey test with FDR = 0.05. Quantification of cells number/field (v) and microscopy images (w) in each well treated with rec.RSPO2. Data shown as mean ± SEM, n = 3 biological replicates. Data was analyzed by one way-ANOVA. Experiment was repeated twice. x–z) After knocking down Lgr4 by siRNA, eP1 cells were treated with rec.RSPO2 (0.5ug/ml) for 24 h. Western blot images (x) and quantification (y) of Beta-Catenin protein in eP1 cells. Beta-actin protein levels were used as loading control. F(3,10)=52.68, P < 0.0001 by one way ANOVA test. Multiple comparison between groups was performed with Tukey FDR = 0.05. Lgr4 mRNA level (z) in eP1 cells 48 h post siRNA transfection. Data shown as mean ± SEM, n = 3-4 biological replicates (x, y), n = 6 independent wells (z). Data analysis was performed using two-tailed Student’s t-test (z). Nuclei were stained with Hoechst 33342 (blue). Scale bars, 100µm. This figure is related to Fig. 3. Source data
Fig 4: Rspo2 inhibits adipogenesis of eP1 cells in vivo.a, b) Microscopy images of H&E staining of matrigel plugs with eP1 cells (a) and eP2 cells (b) without RSPO2 overexpression (top) and with RSPO2 overexpression (lower). Scale bar, 100 µm. Experiment was performed once. c, d) pUC57 plasmid insertion with ApoB and recombined tdTomato sequence. ApoB and recombined tdTomato standard curve generated with plasmid (d). e) Plasma RSPO2 level in CAG-GFP and CAG-Rspo2 ingWAT infection mice, n = 6 mice. Data are presented as mean values + /- SEM. Data analysis was performed using two-tailed Student’s t-test. f) Western blot images of RSPO2 in liver of ingWAT AAV infection mice. HSP90 served as a loading control. g) Immunohistochemistry staining of cleaved caspase-3 in ingWAT. Data are presented as mean values + /- SEM, n = 6 mice. This figure is related to Fig. 4. Source data
Fig 5: other markers expression in subpopulations of adipogenitors.a) Scheme for HFD induced obesity. b) eP3 percentage in ingWAT quantified by FACS. Data show as mean ± SD, n = 6 biological replicates. Data was analyzed using two-tailed Student’s t-test. c) Circulating RSPO2 levels in chow and HFD fed mice. Data shown as mean ± SD, n = 6 biological replicates. Data was analyzed using two-tailed Student’s t-test. d) RSPO2 expression quantified by western blot in ingWAT, visWAT and liver. HSP90 served as loading control. Data show the mean ± SD, n = 6 biological replicates. Data was analyzed by two-tailed Student’s t-test. e) Rspo1-4 mRNA levels in Lin-Sca1+ cells and eP3 cells of ingWAT. Data are presented as mean + /- SEM, n = 4 biological replicates. Data was analyzed by one-way ANOVA, in Lin-Sca1+ group, F(3,12)=602, P < 0.0001; in eP3 group, F(3,12)=326, P < 0.0001. Source data
Supplier Page from CUSABIO Technology LLC for Human R-spondin-2(RSPO2) ELISA Kit