Fig. 5

Activation of TGFβ signaling by R-spondin2. a KEGG pathway analysis of upregulated pathways in R-spondin2-treated cells versus control. b GSEA showing enrichment of TGFβ-associated genes with R-spondin2 treatment. c Heatmap showing genes with altered expression associated with TGFβ and WNT signaling after R-spondin2 treatment (log₁₀FPKM). d Upper panel: Western blot analysis of phosphorylated SMAD2/3 and total SMAD2/3 protein levels in H1 cells at day 2 of differentiation in chemically defined medium treated with different doses of R-spondin2. 0, 10, 20, or 50 ng/mL of R-spondin2 was added at day 0 of hematopoietic differentiation. Lower panel: Densitometric quantitation of Western blot results of phosphorylated SMAD2/3 expression relative to GAPDH and total SMAD2/3 respectively. Results are shown as means ± SD (n = 3). NS, not significant. *P < 0.05. **P < 0.01. (E) Flow cytometry analysis of the percentage of APLNR⁺ cells from control and R-spondin2-treated H1 cells at day 3 of mAGM-S3 co-culture with different doses of SB-431542. R-spondin2 (20 ng/mL) was added along with 0, 1, or 2uM SB-431542 at day 0 of hematopoietic differentiation. Results are shown as means ± SD (n = 3). NS, not significant, **P < 0.01