Fig. 4: BRAFV600E expression in HSPCs induces p16 expression and cell cycle arrest. | Nature Communications

Fig. 4: BRAFV600E expression in HSPCs induces p16 expression and cell cycle arrest.

From: Oncogene-induced senescence in hematopoietic progenitors features myeloid restricted hematopoiesis, chronic inflammation and histiocytosis

Fig. 4

a Representative images of p16 (red) and DAPI (blue) positive cells in BM of mice transplanted with wtBRAF and BRAFV600E-expressing HSPCs at the time of euthanasia. Scale bar: 20 µm. b Quantification of p16+ cells from (A), wtBRAF (blue, n = 3), and BRAFV600E (red, n = 2) groups. Each symbol indicates independent measurements from a given mouse. Lines indicate mean values ± SEM. c Representative images of p16 (red) and DAPI (blue) positive in wtBRAF and BRAFV600E-expressing HSPCs 120 h after in vitro transduction. Scale bar: 50 µm. d Quantification of p16+ cells from (c); wtBRAF (blue) and BRAFV600E (red): n = 4. Lines indicate mean values ± SEM. Statistical test: two-tailed Mann−Whitney. *p < 0.05. e Representative images of 53BP1 foci (red) and DAPI (blue) in wtBRAF and BRAFV600E-expressing HSPCs 120 h post-transduction. f Quantification of 53BP1 foci from (e); wtBRAF (blue, n = 5); BRAFV600E (red, n = 5). Lines indicate mean values ± SEM. Statistical test: Two-tailed Mann−Whitney **p < 0.01 (p = 0.0079). g Growth curves of wtBRAF or BRAFV600E-expressing HSPCs. Cells were counted at the indicated time points after transduction. wtBRAF (n = 8 repeated measurements for four independent donors); BRAFV600E (n = 8 repeated measurements for four independent donors). Data are presented as mean +/− SD. Linear mixed-effects model (LME) followed by post hoc analysis at 48 and 216 h (****p < 0.0001). Inset indicates growth curves of BRAFV600E compared to wtBRAF at early time points. See also Source Data file. h Clonogenic potential of wtBRAF and BRAFV600E-expressing HSPCs in methylcellulose assays over time. wtBRAF (n = 8 repeated measurements for four independent donors); BRAFV600E (n = 8 repeated measurements for four independent donors). Data are presented as mean values +/− SEM. Linear mixed-effects model (LME). ****p < 0.0001. See also Source Data file. All graphs in this figure display data from wtBRAF in blue and from BRAFV600E in red.

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