Fig. 6: Ceramide/PP2A inhibits cytokine-induced AKT, ERK, and JAK/STAT pathways via distinct mechanisms.

a G1E-ER-GATA1 cells were treated with increasing concentrations of DT-061 for 4 h before stimulation with SCF or Epo. Western blotting was conducted to measure SCF and Epo signaling. b Quantitation of blots in (a). p-AKT and p-ERK signals were normalized to their respective protein levels and then normalized to the maximal induction by SCF and Epo, respectively (n = 3 independent experiments). p values were calculated using two-way ANOVA followed by Sidak’s multiple comparisons test. **p = 0.0097. c Cells were treated with increasing concentrations of C6-Cer for 4 h before stimulation with SCF or Epo. Western blotting was conducted to measure SCF and Epo signaling. d Quantitation of blots in (c). p-AKT and p-ERK signals were normalized to their respective protein levels and then normalized to the maximal induction by SCF and Epo, respectively (n = 5 independent experiments). p values were calculated using two-way ANOVA followed by Sidak’s multiple comparisons test. *p = 0.0137. e Cells were treated with increasing concentrations of DT-061 or C6-Cer for 4 h before stimulation with SCF or Epo. Western blotting was conducted to measure JAK2 phosphorylation (n = 3 independent experiments). f Cells were treated with ruxolitinib (Rux) at increasing concentrations before SCF or Epo treatment. Western blotting was conducted to measure SCF and Epo signaling (n = 3 independent experiments). g Cells were treated with 25 μM C6-dhCer or C6-Cer for 30 min before stimulation with SCF or Epo. Western blotting was conducted to measure SCF and Epo signaling. h Quantitation of blots in (g). Phosphorylation signals were normalized to their respective protein levels (n = 3 independent experiments, mean ± SEM). p values were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. n.s., not significant. i Primary human G-CSF-mobilized mononuclear cells were differentiated towards the erythroid lineage. D11 cells were serum-starved, treated with 25 μM C6-dhCer or C6-Cer for 30 min, and stimulated with SCF or Epo. Western blotting was conducted to measure SCF and Epo signaling. j Quantitation of blots in (i). Phosphorylation signals were normalized to their respective protein levels (n = 3 biologically independent samples, mean ± SEM). p values were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. n.s., not significant. k Cells were treated with 10 μM C6-dhCer or C6-Cer for 30 min before stimulation with SCF. AKT and c-KIT phosphorylation was measured by Western blotting. l Quantitation of blots in (k). Phosphorylation signals were normalized to their respective protein levels (n = 4 biologically independent samples, mean ± SEM). p values were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. n.s., not significant. m Cells were treated with 10 μM C6-dhCer or C6-Cer for 30 min before stimulation with Epo. JAK2 and EPOR phosphorylation was measured by Western blotting. n Quantitation of blots in (m). Phosphorylation signals were normalized to their respective protein levels (n = 4 biologically independent samples, mean ± SEM). p values were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. o Model for signaling pathway disruption impacted by acute and prolonged ceramide treatment. Source data are provided as a Source Data file.