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. 2021 May 17;35(10):2936–2947. doi: 10.1038/s41375-021-01275-5

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© The Author(s) 2021

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. 4 — HSPCs cultured for 1 week on GDF-11/RAP-536 pre-treated MDS MSC layers were evaluated by flow cytometry for the expression of A CXCR4 and B CD61. Cumulative data from four experiments are shown as mean ± SD. ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Significance was assessed by two-way ANOVA with Sidak’s and Tukey’s multiple comparisons test. C Representative confocal microscopy images of ITGαVβ3 (green) and CXCR4 (red)/CD45 (green) with nuclear DAPI staining of HSPCs adherent on GDF-11 or GDF-11/RAP-536 pre-treated MDS MSC layers. D Western blot analysis of phospho Smad2/3 (52 kDa) and total Smad2/3 protein (52 kDa) in GDF-11/RAP-536 pre-treated MSCs after co-culture with HSPCs. Duplicate sample aliquots were run on a single gel, which was blotted before being cut into two halves: one being used for phosphor Smad2/3 and one for total Smad2/3 detection. GAPDH (37 kDa) served as reference protein. One representative experiment is shown, N = 3.