Fig. 5: Genetic rewiring of ceramide-cytokine signaling circuit revealed a PP2A-dependent, multi-component mechanism.

a, b G1E-ER-GATA1 cells were pretreated with vehicle or 100 nM okadaic acid (OA) for 1 h followed by 4HPR, C6-dhCer, or C6-Cer treatment for 3 h. Western blotting was conducted to measure p-AKT, p-ERK1/2, and p-STAT5 levels (n = 3 biologically independent samples). Quantitation of blots were shown in Supplementary Fig. 5a and b. c Diagram illustrating the mechanism of SET, Cer, FTY720, and DT-061 to modulate PP2A activity. d CRISPR-Cas9 was used to generate Set^-/- G1E-ER-GATA1 clones. Representative Western blots from 3 independent experiments demonstrated the quantitative depletion of SET protein in two Set^-/- clones. e SCF and Epo signaling in two WT and two Set^-/- clonal lines were measured by Western blotting. f Quantitation of blots in (e). p-AKT, p-ERK, and p-STAT5 signals were normalized to their corresponding protein levels (n = 4 biologically independent samples, mean ± SEM). p values were calculated by two-tailed unpaired Student’s test. g G1E-ER-GATA1 cells were serum starved, treated with 10 μM C6-Cer, FTY720, or DT-061 for 4 h, and stimulated with SCF or Epo. Western blotting was conducted to measure cytokine signaling. h Quantitation of blots in (g). p-AKT, p-ERK, and p-STAT5 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. i WT or Set^-/- cells were treated with increasing concentration of C6-Cer before stimulation with Epo. Two WT and two Set^-/- clones were used. Western blotting was conducted to measure Epo signaling. j Quantitation of blots in (i). Ceramide-dependent inhibition of signaling was assessed by normalizing phosphorylation signals to the maximally induced signal in each cell type (n = 4 independent experiments). Statistical analyses were performed using two-way ANOVA followed by Sidak’s multiple comparisons test, suggesting no significant difference between WT and Set^-/- cells. k Diagram illustrating ORMDL-dependent regulation of ceramide homeostasis and cytokine signaling. l GATA1-dependent regulation of Ormdl1, 2, and 3 mRNA in G1E-ER-GATA1 cells (n = 3 biologically independent samples, mean ± SEM). p values were calculated by two-tailed unpaired Student’s test. m Ormdl3^-/- clones were generated by CRISPR-Cas9. Representative gel of PCR products from 3 independent experiments confirmed the homozygous deletion at Ormdl3 locus. n Representative Western blots from 3 independent experiments showing that ORMDL proteins were quantitatively reduced in two Ormdl3^-/- clones after erythroid differentiation. o WT and Ormdl3^-/- cells were serum-starved and stimulated with Epo at increasing concentrations. Two WT and two Ormdl3^-/- clonal lines were used. Epo signaling was measured by Western blotting. p Quantitation of blots in (o). Phosphorylation signals were normalized to their corresponding protein levels (n = 4 independent experiments). p values were calculated using two-way ANOVA followed by Sidak’s multiple comparisons test. ***p < 0.0001. Source data are provided as a Source Data file.