Fig. 4: Role of S1PR1 in cP1P-induced HIF1α translation.

a, b UCB-MSCs were pretreated with cycloheximide (10 μM) for 30 min prior to cP1P (1 μM) treatment for 24 h. a The protein expressions of HIF1α and β-Actin were detected by western blotting. n = 3. b Cells were immunostained with HIF1α -specific antibody. n = 10–12. Scale bars are 8 μm. Magnification × 1,000 (left panel). c, e UCB-MSCs were treated with cP1P (1 μM) for 24 h. c HIF1A mRNA expression level was normalized by 18 S rRNA expression level. n = 6. d UCB-MSCs were incubated with cP1P (1 μM) for 0, 6, 12, or 24 h. The protein expression of prolyl hydroxylated HIF1α, HIF1α, and β-Actin were detected by western blotting. n = 3. e Co-immunoprecipitation of VHL and α-Tubulin with IgG and HIF1α antibodies are shown in left panel. Total protein expressions of HIF1α, VHL, and α-Tubulin in lysates are shown in right panel. n = 4. f UCB-MSCs were pretreated with JTE013 (10 μM) or VPC23019 (1 μM) for 30 min prior to cP1P (1 μM) treatment for 24 h. The protein expressions of HIF1α and β-Actin were detected by western blotting. n = 4. g–i UCB-MSCs were transfected with S1PR1, S1PR3, or NT siRNA for 24 h prior to cP1P (1 μM) treatment for 24 h. g HIF1 activities were assessed by dual luciferase reporter assay. n = 4. h The protein expressions of HIF1α, S1PR1, and β-Actin were detected by western blotting. n = 3. i UCB-MSCs were immunostained with HIF1α-specific antibody. n = 6. Scale bars are 8 μm. Magnification × 1,000 (left panel). All blot and immunofluorescence images are representative. Quantitative data are presented as a mean ± S.E.M with scatter plots. * indicates p < 0.05