Skip to main content
. 2023 Nov 18;6:1174. doi: 10.1038/s42003-023-05540-4

Fig. 5. TC expression in endothelial cells results in oncogene induced senescence, which can be overcome by a secondary mutation.

Fig. 5

a Representative images showing endothelial cells that were left untreated or incubated with dox for 24 h to induce TC expression, then stained for DAPI (nuclei; blue), p16 (magenta), and FLAG (TC; yellow). Imaging was performed on a Zeiss fluorescence widefield microscope using a 40x objective. Scale bars = 25 μm. b Fluorescence intensity of p16 staining as shown in (a), to quantify its expression, n = 3. Statistical significance was determined with an unpaired t-test. c Representative images of GFP (TC) high, low, negative and uninduced populations stained for SA-β-gal activity, 4 days after addition of dox. d Frequency of cells positive for SA-β-gal activity in GFP high, GFP low, GFP-, and dox- endothelial cell populations. A minimum of 150 cells per condition were analysed, n = 3. Significance was calculated with a one-way ANOVA and Tukey’s multiple comparisons test. e Representative images showing the formation of GFP+ foci, whereby GFP is a marker for TC expressing endothelial cells, after 10 days exposure to dox. Scale bars = 200 μm, n = 3. f Fold change of TC high population between days 7 and 14 after nucleofection with control and Cdkn2a knockout guide RNA, n = 4. Statistical significance was calculated with a two-way ANOVA and Sidak’s multiple comparisons test. g Normalised cell number in Cdkn2a knockout endothelial cells, treated with dox or not, over 14 days, n = 4. Statistical significance was calculated with a two-way ANOVA and Sidak’s multiple comparisons test. All P-values are outlined in Supplementary Table 1. h Fold change of Ki67 expression within the TC high population between days 7 and 14 after addition of dox in comparison to uninduced endothelial cells, n = 4. Statistical significance was calculated with a two-way ANOVA and Sidak’s multiple comparisons test. i Representative images showing the formation of GFP+ foci, whereby GFP is a marker for TC expressing endothelial cells, after Cdkn2a knockout. Scale bars = 200 μm, n = 4. In all panels error bars show SEM, and *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.