Fig. 4

H₂O₂ controls Shh secretion. (A-D) DiO RGC labelling at 3 dpf in control embryos (A), embryos treated with Nox-i (B), Nox-i+SAG (C) or SAG (D) from 2 to 3 dpf. The maximum length of the projections was quantified (E). The n values are indicated at the bottom of each column of the graph. Representative images are shown. (F) HyPer imaging in the glucose oxidase (Gox)-treated HeLa-HyPer cells. Scale bar, 20 µm. (G) Quantification of the H₂O₂ levels in the control and glucose oxidase-treated HeLa-HyPer cells. Control: n=10; Gox: n=15. (H) Western blot analysis of Shh in the cell lysates (C) or culture medium (M) upon glucose oxidase treatment. (I) Quantification of the Shh precursor following Gox treatment. Shh-precursor (Gox)/Shh-precursor (control). (J) Quantification of the amount of N-terminal Shh peptide in the medium (cf Experimental Procedures) following Gox treatment. The quantifications were performed on 5 independent experiments; a representative western blot is shown in H. (K, L) Shh-GFP fluorescence subcellular distribution in control or Gox-treated cells. Scale bar, 10 µm. (M) Shh-GFP (green) colocalized with the BODIPY TR ceramide Golgi marker (red) in Gox-treated cells. The error bars represent the standard error of the mean (SEM) (*p<0.05; **p<0.01; ***p<0.001).