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. 2021 Jul 27;12:4559. doi: 10.1038/s41467-021-24876-1

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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. 6 — a Experimental strategy adopted to dissect p16 and SASP induction. Relative mRNA expression of (b) p16 and (c) p21 upon treatment with scrambled (scr) shRNA or shRNA against p16/ARF (shp16). Data are presented as mean +/− SEM. Each dot represents an independent donor (n = 4). Statistical test: Kruskal−Wallis with Dunn’s multiple comparison. d Growth curves of wtBRAF or BRAF^V600E-expressing HSPCs depleted or not for p16. n = 4 independent donors. Data are presented as mean +/− SD. Statistical test: Kruskal-Wallis with Dunn’s multiple comparison at 48 and 216 h. Inset indicates growth curves at early time points. e Percentage of GFP⁺ cells of wtBRAF or BRAF^V600E-expressing HSPCs depleted or not for p16. Data are presented as mean +/− SD. Each dot represents an independent donor (n = 4). Statistical test: Kruskal−Wallis with Dunn’s multiple comparison. f Clonogenic potential upon treatment with scr or shp16, in methylcellulose assays at 120 h post transduction, wtBRAF: n = 12 repeated measurements for four independent donors; BRAF^V600E: n = 12 repeated measurements for four independent donors. Statistical test: Two-tailed Mann−Whitney. g–k Gene expression analysis of SASP cytokines (IL1α, IL1β, IL8, TNFα, and MCP1) in wtBRAF or BRAF^V600E-expressing cells upon treatment with scr or shp16. Data are presented as mean +/− SEM. Each dot represents an independent donor transduced with BRAF^V600E (N = 4). Statistical test: Two-tailed Mann−Whitney. l Clonogenic potential of wtBRAF and BRAF^V600E-expressing HSPCs upon treatment with scrambled shRNA (scr) or shRNA against RELA/p65 (shp65) in methylcellulose assays at 120 h post transduction with wtBRAF or BRAF^V600E vectors. n = 12 repeated measurements for four independent donors in each group. Data are presented as mean +/− SEM. Statistical test: Two-tailed Mann−Whitney. All graphs in this figure display data from: wtBRAF treated with scr in dark blue or with shRNA either against p16 in light blue; BRAF^V600E treated with scr in dark red or with shRNA either against p16 in light red. Gene expression values are represented as 2^^−ΔCt relative to housekeeping gene. *p < 0.05; **p < 0.01; ****p < 0.0001.