Fig. 2: Ceramide homeostasis is required for erythroblast survival and differentiation. | Nature Communications

Fig. 2: Ceramide homeostasis is required for erythroblast survival and differentiation.

From: A transcriptional network governing ceramide homeostasis establishes a cytokine-dependent developmental process

Fig. 2

a G1E-ER-GATA1 cells were treated with DMSO or 2 μM 4HPR for 24 or 48 h and Cer and dhCer levels were quantified by lipidomics (n = 4 biologically independent samples, mean ± SEM). The levels of lipids were normalized to the level of inorganic phosphate (Pi). p values were calculated by two-tailed paired Student’s test. b Heatmaps depicting levels of individual species of Cer and dhCer from (a). c, d G1E-ER-GATA1 cells were treated with DMSO or 4HPR in the absence or presence of β-estradiol. Left, live cell numbers were quantified at 0, 12, 24, 36, and 48 h after treatment by Trypan Blue dye exclusion assay (n = 4 biologically independent samples). p values were calculated using two-way ANOVA followed by Sidak’s multiple comparisons test. Right, viability was calculated by normalizing live cell numbers of 4HPR-treated samples to DMSO-treated ones at each time point (n = 4 biologically independent samples, mean ± SEM). p values were calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. *p = 0.0319, ***p < 0.0001. e Primary human G-CSF-mobilized mononuclear cells were differentiated towards the erythroid lineage. At D9, cells were treated with DMSO or 4HPR. Left, live cell numbers were quantified at 0, 24, 48, 72, and 96 h after treatment by Trypan Blue dye exclusion assay (n = 3 biologically independent samples). p values were calculated using two-way ANOVA followed by Sidak’s multiple comparisons test. Right, viability was calculated by normalizing live cell numbers of 4HPR-treated samples to DMSO-treated ones at each time point (n = 3 biologically independent samples, mean ± SEM). p values were calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. ***p < 0.0001. f Representative picture showing impaired hemoglobinization of 4HPR-treated cells after differentiation. g G1E-ER-GATA1 cells were treated with β-estradiol with or without 4HPR, and mRNA levels were quantified by RT-qPCR (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.

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