Fig. 1

DNA methylation mediates the change of chitin degradation in Bombyx mori wing. a Dot blot analysis (left) and immunofluorescent staining (right) of DNA methylation levels 48 h after treatment of newly molted pupae with 5-aza-dC. Dot blot was conducted using 5mC antibody (top left) and then the quantitative analysis of relative blot intensity (5mC/Input) was calculated automatically using ImageJ software (bottom left). 5mCs were stained with secondary antibody (right, red) and the nuclei were stained with DAPI (right, blue). The relative fluorescence intensity of 5mCs was calculated using ImageJ software (bottom right). The images were captured by a laser confocal microscope. Scale bar: 40 μm. 5mC: 5-methylcytosine. DAPI: 4′, 6-diamidino-2-phenylindole. b A newly molted pupa (P0 stage) was injected with 20 μg 5-aza-dC- or control ddH₂O. Phenotype changes at adult stage were captured by stereomicroscope. Scale bar: 5000 μm (left). Phenotype differences between the wing discs of 5-aza-dC- or ddH₂O-treated silkworm at different pupal developmental stages were captured by stereomicroscope. Scale bar: 2000 μm. Pn: n-day-old of pupae. A: Adult. c Chitin from the pupal wing discs or adult wings of 5-aza-dC- or ddH₂O-treated silkworm at P6-A stages were extracted for chitin content assays using spectrophotometry. d Chitin staining of the crosscut wings of 5-aza-dC- or ddH₂O- treated silkworm at P6-A stages. Chitin was stained with Fluorescent Brightener 28 (blue) and the nuclei were stained with propidium iodide (red). The images were captured by a laser confocal microscope. Scale bar: 40 μm. Each data point is the mean ± SE of three independent assays. For the t test: p < 0.05 (*) or p < 0.01(**)