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Fig. 7
Defects in endocytic trafficking is responsible for PIK3C3 deficiency-induced polarity defect. a Western blot analysis of LC3B in dissected gut. MEF lysate (total cell lysate of mouse embryonic fibroblasts under autophagic conditions) was used as the positive control. The LC3B antibody can detect the activated form of zebrafish LC3B (LC3B-II) but not LC3B-I. b qRT-PCR analysis for the efficiency of siRNA to UVRAG or ATG14 in Caco2 cells. ACTB is the internal control and data represent mean ± SD from three biological repeats (*p < 0.05; **p < 0.01 by unpaired two-tailed Student’s t-test). c, d UVRAG but not ATG14 knockdown induces abnormal lumen formation and E-Cadherin mis-localization in Caco2 cyst. Scale bar, 10 µm. Data represent mean ± SD of the percentages of abnormal cyst (two or multiple lumens) from three biological repeats. NS, non-significant; ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparison. e qRT-PCR analysis of ER stress gene hspa5 (heat shock protein family A member 5) in 8 dpf embryos. gapdh is the internal control and data represent mean ± SD from three independent repeats. p-value determined by unpaired two-tailed Student’s t-test (**p < 0.01). f Immunofluorescence staining of gut cross sections. In the pik3c3 mutant, HSPA5 signal is detected in liver (arrow) and a few cells in gut. Treatment of WT embryos with tunicamycin (an ER stress inducer, 1 µM from 7 to 8 dpf) strongly induces HSPA5 in liver and gut. Scale bar, 50 µm