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. 2018 Dec 13;26(9):1796–1812. doi: 10.1038/s41418-018-0245-x

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Impact of TNFα/SMase/ceramide pathway on EpoCD34/HSPCs differentiation. a May–Grünwald–Giemsa staining of CD34/HSPCs at day 0 and EpoCD34/HSPCs at day 9 of treatment observed with a ×40 objective. Each picture is representative of three independent experiments. Enucl/Ret: enucleation/reticulocyte; E: erythroblast; Ortho: orthochromatophilic. b Representation of the different cell types distribution on MGG-stained slides. c Microscope observation (×20) of distinctive morphological aspects of hematopoietic colonies obtained after EpoCD34/HSPCs treatments at day 14. E: erythroid; GM: granulocyte–monocyte; GEMM: granulocyte–erythrocyte–monocyte–megakaryocyte; G: granulocyte; M: monocyte/macrophage. d Representation of the different colonies distribution in colony formation assays of EpoCD34/HSPCs treatments at day 14. e Flow cytometry analysis of CD11b expression in EpoCD34/HSPCs from day 0 to 9 of culture. f NBT staining with GW4869 and si-SMPD3 at 96 h. g Real-time PCR analysis of MMP-9 expression with GW4869 and si-SMPD3 at 96 h. h Western blot analysis of GATA-1, GATA-2, and PU.1 in EpoCD34/HSPCs after 96 h of treatment. β-Actin expression was used in Western blot analyses as an internal control and was revealed each time on the same membrane than one or several proteins of interest. i Real-time PCR analysis of miR-144, -451, -155, -146a, and -223 expression levels in EpoCD34/HSPCs. Bars represent the mean ± SD of five independent experiments. One-way ANOVA with repeated measures followed by Dunnett’s post hoc test or two-way ANOVA followed by Holm–Sidak’s post hoc test show the statistical significance *^,$P < 0.05, **P < 0.01, ***P < 0.001 (*: treatment vs. untreated, $: treatment vs. TNFα)

Fig. 2 — Impact of TNFα/SMase/ceramide pathway on EpoCD34/HSPCs differentiation. a May–Grünwald–Giemsa staining of CD34/HSPCs at day 0 and EpoCD34/HSPCs at day 9 of treatment observed with a ×40 objective. Each picture is representative of three independent experiments. Enucl/Ret: enucleation/reticulocyte; E: erythroblast; Ortho: orthochromatophilic. b Representation of the different cell types distribution on MGG-stained slides. c Microscope observation (×20) of distinctive morphological aspects of hematopoietic colonies obtained after EpoCD34/HSPCs treatments at day 14. E: erythroid; GM: granulocyte–monocyte; GEMM: granulocyte–erythrocyte–monocyte–megakaryocyte; G: granulocyte; M: monocyte/macrophage. d Representation of the different colonies distribution in colony formation assays of EpoCD34/HSPCs treatments at day 14. e Flow cytometry analysis of CD11b expression in EpoCD34/HSPCs from day 0 to 9 of culture. f NBT staining with GW4869 and si-SMPD3 at 96 h. g Real-time PCR analysis of MMP-9 expression with GW4869 and si-SMPD3 at 96 h. h Western blot analysis of GATA-1, GATA-2, and PU.1 in EpoCD34/HSPCs after 96 h of treatment. β-Actin expression was used in Western blot analyses as an internal control and was revealed each time on the same membrane than one or several proteins of interest. i Real-time PCR analysis of miR-144, -451, -155, -146a, and -223 expression levels in EpoCD34/HSPCs. Bars represent the mean ± SD of five independent experiments. One-way ANOVA with repeated measures followed by Dunnett’s post hoc test or two-way ANOVA followed by Holm–Sidak’s post hoc test show the statistical significance *^,$P < 0.05, **P < 0.01, ***P < 0.001 (*: treatment vs. untreated, $: treatment vs. TNFα)