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Fig. 5 Smad-reactive oxygen species pathway mediates the killing of unfit cells. a, d Shh-unfit cells activate the Smad2/3/4-dependent reporter gene (SBE-Luc) (a) and reactive oxygen species (ROS) production (d). Confocal images show whole-mount fluorescent in situ hybridisation of luciferase mRNA (a) and immunostaining for 8-OHdG (d) (magenta) in mosaic larvae expressing membrane GFP alone or with SmoCA or Ptch1 (green). Scale bar = 10 ?m. In a, the luciferase intensity of each GFP+ cell is plotted. Two-tailed one-way ANOVA was used. In d, violin plots show the 8-OHdG intensity of each GFP+ cell. Two-tailed one-way ANOVA was used. b, c, e, f, h, i Smad3bDN overexpression (b, c), ROS inhibition (e, f), and Bcl2a overexpression (h, i) blocked SmoCA- or Ptch1-expressing cell apoptosis. Confocal images show whole-mount immunostaining of active caspase-3 (grey) in mosaic larvae expressing mKO2 with SmoCA or Ptch1 (magenta), injected with GFP or GFP-Smad3bDN (b, c), treated with D2W (control) or N-acetyl-l-cysteine (NAC, a ROS scavenger) (e, f), or injected with GFP or GFP-Bcl2a (h, i). Scale bar = 10 ?m. The graphs on the right show the mean + SEM of mKO2+ (SmoCA, Ptch1) and caspase-3-active cell frequencies. An unpaired two-tailed t-test was used for the statistical analysis. g Schematic diagram showing the elimination of Shh-unfit cells. Source data are provided as a Source Data file.