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. 2020 Oct 8;11:5061. doi: 10.1038/s41467-020-18900-z

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© The Author(s) 2020

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 — a Western blotting for H3K27me3 in the indicated OSKM-reprogrammed P2 MEFs and quantification normalized to H3 (upper panel), and the statistics for the quantification (lower panel). b Schematic depiction of JMJD3 and a catalytic mutant JMJD3 (JMJD3 mut). c Western blotting for H3K27me3 and FLAG (exogenous FLAG-tagged JMJD3 constructs) in OSKM-reprogrammed P2 MEFs at day 5 and H3K27me3 quantification normalized to H3 (left panel) and statistics for the quantification (right panel). d Number of GFP⁺ colonies at day 11 in OSKM-reprogrammed P2 MEFs with the indicated JMJD3 constructs. e Number of GFP⁺ colonies at days 11 (with Vc) and 16 (without Vc) in OSKM-reprogrammed P2 MEFs with shRNAs against firefly Luciferase (shLuc) or Jmjd3 (shJmjd3; two independent shRNAs). f Number of GFP⁺ colonies at day 11 in OSKM-reprogrammed P2 MEFs with shLuc or shJmjd3, together with the indicated JMJD3 rescue mutants or Empty. g Phase contrast and NANOG immunofluorescent images of typical colonies (left panel) and number of NANOG⁺ colonies in OSKM-reprogrammed WT and Jmjd3 KO P2 MEFs with the indicated constructs (right panel) at day 11. Scale bars, 100 μm. h Western blotting for H3K27me3 in OSKM-reprogrammed WT and Jmjd3 KO P2 MEFs with the indicated constructs at day 5, H3K27me3 quantification normalized to H3 (left panel) and statistics for the quantification (right panel). Error bars represent the s.e.m. Data are presented as mean ± s.e.m. from n = 3 a, c, d–h biologically independent experiments. Statistical analyses were performed using a two-tailed unpaired Student’s t-test c–e, g, h or one-way ANOVA followed by a Holm–Sidak multiple comparison test a, f (*P < 0.05; **P < 0.01; ***P < 0.001). P values: 0.0198 (D5 + Vc − JMJD3 vs. D5 + Vc + JMJD3), 0.0001 (D5 − Vc − JMJD3 vs. D10 − Vc − JMJD3), 0.0001 (D10 + Vc − JMJD3 vs. D10 + Vc + JMJD3) a; 0.0059 c; 0.0031 d; 0.0135, 0.0009, 0.0048, 0.0023 e; 0.0255 (shLuc + Empty vs. shJmjd3 #1 + Empty), 0.0001 (shJmjd3 #1 + Empty vs. shJmjd3 #1 + Mut #1), 0.0065 (shLuc + Empty vs. shJmjd3 #2 + Empty), 0.0001 (shJmjd3 #2 + Empty vs. shJmjd3 #2 + Mut #2) f; 0.0018, 0.0009 g; 0.0053, 0.0189 h. Source data are provided as a Source Data file.