Loss of intestinal epithelial Shh signaling leads to an increase in ER stress and a decrease in autophagy in intestinal epithelial cells (IECs).

<p>Transmission electron microscopy revealed a dilated ER lumen in <i>Shh</i>^ΔIEC Paneth cells (black arrows, B) when compared to controls (black arrowheads, A). Ultrastructural examination revealed granule abnormalities in mutant mice (B). Paneth granules were counted in both <i>Shh</i>^ΔIEC and control mice and no significant modulation was found in the number of granules per Paneth cell between control and mutant mice (C) (n = 4). The mean size of Paneth cell granules was determined using MetaMorph software and statistical analysis revealed that granules were significantly smaller in <i>Shh</i>^ΔIEC ileum when compared to controls (D) (n = 4). Western blot analysis against defensin 4, IRE1α, p62, lipidated LC3b-II and non-lipidated LC3b-I proteins were performed on ileal extracts from control and <i>Shh</i>^ΔIEC mice (E). Densitometry analysis of exposed films using ImageJ revealed a significant 3.85-fold decrease in defensin 4 expression (F) a significant 3.6-fold increase in IRE1α (G) and a significant 2.13-fold increase in p62 (H) in <i>Shh</i>^ΔIEC mice compared to controls (n = 3). An 11-fold decrease in lipidated LC3b-II to non-lipidated LC3b-I ratio in <i>Shh</i>^ΔIEC mice can be seen compared to controls (I) (n = 3). Two-way ANOVA ***<i>p</i><0.001 (C–D). Student t-test *<i>p</i><0.05, **<i>p</i><0.01 (F–I). Error bar represents SEM. Scale bar: 2 µm. Def 4, defensin 4.