lkb1 mutant larvae show impaired activation of autophagy following yolk depletion. (A) Representative Western blot analysis of Lc3-II, Atg5, p62 and Tubulin (loading control) in total protein lysates of wt and lkb1 trunks between 5–7 dpf. Larvae were treated with chloroquine (2.5 μM) for 14 h prior to processing. The marked decrease in Lc3-II and Atg5-containing complexes together with the p62 accumulation indicate impaired autophagy in lkb1 larvae. Uncropped images of the blots are shown in Supplementary Fig. S7A–C. (B–E) Transverse vibratome sections (150 μm) of intestine of 7 dpf wt and lkb1 mutants stained with anti-LC3B antibody (green), rhodamine-phalloidin to detect F-actin (red) and DAPI to detect nuclei (blue). Lc3B staining in the lkb1 intestine is barely detectable (C,E) and more foci of intense staining were visible in wt sections compared to sections from lkb1 mutants. (F,G) Immunohistochemical analysis of transverse paraffin sections (5 μm) of liver and intestine of 7 dpf wt and lkb1 larvae reveals high levels of p62 accumulation in lkb1 liver and intestine. Magnification: 40×. PD: pronephric ducts; L: liver; SI: intestine.
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