Fig. 4: Ceramide homeostasis commissions cytokine signaling in human and murine erythroblasts.

a–c G1E-ER-GATA1 cells were treated with β-estradiol (E2) for 24 h, serum-starved, treated with 2 μM 4HPR for 4 h, and stimulated with SCF or Epo for 5 min. SCF and Epo signaling were measured by Western blotting (b). Quantitation of p-AKT, p-ERK, and p-STAT5 is shown in (c). Values were normalized to their respective protein levels (n = 4 independent experiments, mean ± SEM). p values were calculated by two-tailed paired Student’s test. d–f G1E-ER-GATA1 cells were treated with β-estradiol for 24 h, serum-starved, treated with 25 μM C6-dhCer or C6-Cer for 4 h, and stimulated with SCF or Epo for 5 min. SCF and Epo signaling were measured by Western blotting (e). Quantitation of p-AKT, p-ERK, and p-STAT5 signals is shown in (f). Values were normalized to their corresponding protein levels (n = 4 independent experiments, mean ± SEM). p values were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. g–i Primary human erythroblasts at D11 were serum-starved, treated with 2 μM 4HPR, 25 μM C6-dhCer, or 25 μM C6-Cer for 4 h, and stimulated with SCF or Epo for 5 min. SCF and Epo signaling were measured by Western blotting (h). Quantitation of p-AKT, p-ERK, and p-STAT5 signals is shown in (i). Values were normalized to their corresponding protein levels (n = 4 independent experiments, mean ± SEM). p values were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. j, k G1E-ER-GATA1 cells were treated with 4HPR for 4 h and cytokine-dependent phosphorylation of receptors (KIT and EPOR) were measured by Western blotting. Values were normalized to their corresponding protein levels (n = 3 biologically independent samples, mean ± SEM). p values were calculated by two-tailed paired Student’s test. l, m G1E-ER-GATA1 cells were treated with C6-dhCer or C6-Cer for 4 h and cytokine-dependent phosphorylation of receptors (KIT and EPOR) were measured by Western blotting. Values were normalized to their corresponding protein levels (n = 4 biologically independent samples, mean ± SEM). p values were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. Source data are provided as a Source Data file.