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. 2017 Jul 20;7:6089. doi: 10.1038/s41598-017-06470-y

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© The Author(s) 2017

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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Inhibition of TGF-β signaling supports self-renewal of mouse primary epidermal progenitor cells in serial 3T3-J2 co-culture. (a) Rhodamine B staining of newborn mouse-derived, RepSox-expanded P1 epidermal cells grown in 3T3-J2 co-culture in the presence or absence of 1 μM RepSox for 14 days. Bar = 10 mm. (b) Distribution of epidermal clone sizes at day 14. Data shown are mean ± s.e.m. (n = 3). *P < 0.05; **P < 0.01. (c–f) Removal of RepSox associates with nuclear translocation of Smad2/3 in newborn mouse epidermal cells in 3T3-J2 co-culture. (c) Experimental scheme. Mouse primary epidermal keratinocytes were grown in 3T3-J2 co-culture in the presence of 1 μM RepSox. Subsequently, the cultures were maintained in the presence or absence of 1 μM RepSox for 48 hrs before fixation for immunohistochemistry. (d) A representative image of mouse primary keratinocyte clones in 3T3-J2 co-culture. Note that epidermal cell clones show a cobblestone appearance with a distinct clone border (dotted line). Epi, epidermal cell clone. Bar = 50 μm. (e) Representative immunofluorescence images of mouse epidermal cells without (upper) or with (lower) removal of RepSox, stained with anti-Smad2/3 antibodies (left panels) and counterstained with Hoechst 33342 (middle panels). Right panels show merged images. Bar = 10 μm. (f) Nuclear-to-cytoplasmic (N/C) ratios of Smad2/3 as determined by fluorescence intensity. Data shown are mean ± s.e.m. (n = 10). *P < 0.001. (g) Immunohistochemistry of representative epidermal cell clones grown in 3T3-J2 co-culture for 14 days in the presence of 1 μM RepSox, stained with anti-p63 (upper) and anti-CK14 (middle) antibodies. The lower panel represents a control clone stained with the secondary antibodies alone. Data shown are peripheral regions of three different epidermal clones with similar diameter. Bar = 100 μm. (h) Distribution of epidermal clone sizes in serial co-culture with 3T3-J2 cells in the presence of 1 μM RepSox.