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. 2019 Dec 6;10:5576. doi: 10.1038/s41467-019-13314-y

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© The Author(s) 2019

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 — CSB binds to the p21^Waf1 promoter. a PCR (agarose gel) and b quantitative PCR (histogram) analyses of several DNA fragments in the p21^Waf1 promoter from ChIP assays with either N-term α-CSB [Bethyl] or C-term α-CSB [Abcam] antibodies. c Scheme not at scale of primer positions on the p21^Waf1 promoter and summary of positive amplifications in turquoise (boxes in magenta indicate no amplification). n = 3 independent experiments; one-way ANOVA (5’p53RE: F = 7.698, DFn = 2, DFd = 6, p = 0.0220. 3’p53RE: F = 17.11, DFn = 2, DFd = 6, p = 0.0033; TATA: F = 0.8699, DFn = 2, DFd = 6, p = 0.4659) with post-hoc Tukey’s test for each tested regions vs. Mock. d Scheme of the CDKN1A (p21^waf1) promoter-eGFP reporter plasmid. e Representative confocal acquisitions of cells stably expressing the CDKN1A (p21^waf1) promoter-eGFP reporter upon shCSB#2 silencing or control shSCR, immunostained for GFP (green) to increase the signal of endogenous GFP, and counterstained with Hoechst (blue) after maximum intensity projection with Imaris; scale bar = 50 µM, and f quantification of the GFP mFI/cell; n = 127 cells/condition from three independent experiments, mean ± SEM; unpaired t-test (two-tailed) (t = 4.555, DF = 253), p-value vs. shSCR. g WB of GFP and GAPDH (loading control) in cells not expressing (Mock GFP) or stably expressing (Mock shRNA) the CDKN1A (p21^waf1) promoter-eGFP reporter, not silenced (shSCR) or silenced for CSB (shCSB#2). Cells that express GFP display a band at 27 kDa, and also an upper band at around 60 kDa, in particular upon CSB silencing, corresponding to dimers observed in other conditions⁷⁰. Source data are provided as Source Data files.