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. 2018 Apr 18;9:1531. doi: 10.1038/s41467-018-03876-8

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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. 3 — OSM treatment enhances stem cell serial transplantation and inhibits differentiation. a FACS analysis of stem cells reisolated from primary transplant recipients receiving either control (left), FGF (middle), or OSM-treated (right) stem cells. Only the live, CD11b⁻Sca1⁻CD31⁻CD45⁻CD34⁺α7 integrin⁺ stem cell population is shown. b MuSCs (CD11b^-Sca1^-CD31^-CD45^-CD34⁺α7 integrin⁺GFP⁺) were reisolated from OSM primary transplant mice as described in a, transplanted equally into 5 limbs (secondary transplant recipients; 50 MuSC/limb; 8–12 week old recipients), and monitored by serial BLI. At day 25 following transplant, cardiotoxin (CTX) was injected to induce myofiber damage and stimulate stem cell expansion and tissue regeneration. The average radiance at each timepoint is indicated, and demonstrates regenerative capacity (BLI signal loss followed by rebound) of serially transplated OSM-treated MuSC. Percentage of engrafted mice was 40% both pre and post-CTX injury (2/5 total mice). Note that untreated cells give no engraftment, and no MuSC are therefore present for reisolation/transplant. c qRT-PCR analysis of Pax7 expression following 6 day culture of FACS-sorted MuSC in the presence of OSM (gray circles) or buffer control (black). Mean±SD is shown, n = 3 replicates. d qRT-PCR analysis of myogenin expression following 6 day culture of FACS-sorted MuSC with the indicated cytokines and growth factors. Expression was normalized to GAPDH expression. Mean±SD is shown, n = 3 replicates. e Timecourse intracellular flow cytometry of primary mouse myoblasts treated for the indicated period of time with OSM, and stained for either phospho-STAT3 (left) or phospho-STAT5 (right). 0 (unstimulated), 15, 30, and 45 min timepoints are shown following cytokine addition. Frequencies are represented as histograms. f ChIP performed using primary myoblasts cultured for 6 days either with or without OSM. Immunoprecipitation was performed with either control IgG or STAT3-specific antibodies, and PCR was performed using primers specific for a predicted STAT3 binding site adjacent to the Pax7 locus