Fig. 2. Newly generated oncogenic cells undergo cellular senescence.

a–d Representative confocal images show cells with GFP (a, d) or mCherry (b, c) alone (control), or with Ras^G12V, TP53^R175H, or Ras^G12V and TP53^R175H (GFP⁺, mCherry⁺, Ras⁺, TP53^RH+, or Ras⁺TP53^RH+ cells) (green in a, d, magenta in b, c). Scale bar: 20 μm. In a, arrows indicate γH2AX (magenta)-accumulated GFP⁺ oncogenic cells. Violin plots of γH2AX intensities in GFP⁺ cells show 75th, 50th (median), and 25th percentiles. In b, SA-β-gal was visualised by SPiDER-βGal (green). Arrowheads indicate SA-β-gal-expressing mCherry⁺ oncogenic cells, and arrows indicate cells SA-β-gal-expressing neighbouring cells. Violin plots of SA-β-gal intensities in mCherry⁺ cells show 75th, 50th (median), and 25th percentiles. In c, EdU⁺ cells (green) were observed in mCherry⁺ or TP53^RH+ cells, but not Ras⁺ or Ras⁺TP53^RH+ cells (magenta). Yellow broken lines: nuclei of mCherry⁺ cells. Bar plots show EdU⁺mCherry⁺ cells (mean ± SEM). Each dot represents one larva. A two-tailed one-way ANOVA test with Sidak correction (a, b, c) was used. In d, fluorescent in situ hybridization visualised cdkn2a/b mRNA (magenta). Arrows or arrowheads indicates cells expressing cdkn2a/b weakly or strongly, respectively. Bottom graphs show percentages of GFP⁺ cells with weak or strong cdkn2a/b expression. Fisher’s exact test with Bonferroni correction was used. e Representative confocal images show cells expressing mKO2⁺Ras^G12V (Ras⁺) (magenta) and nucleus (upper: blue, lower: grey). Skin cells visualised with krt4p:gal4; UAS:EGFP (green). Scale bar: 20 μm. Box plots of estimated nuclear or cytoplasmic size (μm³) show first and third quartile, median is represented by a line, whiskers indicate the minimum and maximum. Each dot represents one cell. Unpaired two-tailed t-test was used.f Representative images show larvae with GFP⁺Ras^G12V (Ras⁺) cells (green) with control MO or cdkn2a/b + tp53 MOs. Scale bar: 200 μm. Box plots of relative Ras⁺ cell number between 2/1 dpf ratio show first and third quartile, median is represented by a line, whiskers indicate the minimum and maximum, and outliers are shown as dots outside of the box. Each dot represents one larva. Unpaired two-tailed t-test was used. Source data are provided as a Source Data file.