Extended Data Fig. 3: Selection and identification of Aregs’ effectors.
From: Identification of a regulatory pathway inhibiting adipogenesis via RSPO2
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.
