Enteroids derived from Myo5b KO mice form inclusions via apical bulk endocytosis. (A) H&E staining and TEM of enteroids derived from the duodenum of neonatal control and Myo5b KO mice. Morphologically, enteroids from control and Myo5b KO appeared similar; however, TEM revealed the presence of large intracellular inclusions lined with microvilli in Myo5b KO enteroids. Immunofluorescence staining showed the presence of numerous P-ERM–positive (red) inclusions in Myo5b KO–derived enteroids. H&E scale bars = 50 µm, TEM scale bars = 500 nm, immunofluorescence micrograph scale bar = 50 µm. (B) Enteroids generated from Myo5b KO mice were treated with DMSO (vehicle), Dyngo, EIPA, and cyclosporin to determine the mechanism of inclusion formation. P-ERM (red) and DPPIV (green) staining showed the presence of numerous inclusions, some still contiguous with the apical membrane in Myo5b KO enteroids. Arrows indicate the positions of inclusions. (C) The number of inclusions attached to the apical membrane and the total number of inclusions present in individual enteroids was quantified. The percentage of inclusions associated with the brush border in enteroids was then calculated, and the percentage of inclusions contiguous with the apical membrane in each individual enteroid was plotted. With administration of Dyngo, there was a significant increase in the percentage of inclusions that were still attached to the apical membrane compared with DMSO-treated enteroids. (D) The total number of inclusions was quantified in individual Myo5b KO enteroids to address whether treatments (Dyngo, EIPA, or cyclosporin) impacted inclusion formation. Administration of cyclosporin resulted in a significant decrease in inclusion formation in Myo5b KO enteroids. Scale bars = 50 µm for left panel of B, 10 µm for right panel of B. Enteroids were generated from three to five control and Myo5b KO mice. n = 6–15 enteroids analyzed per treatment; *, P < 0.05. One-way ANOVA was performed with the Bonferroni post hoc test for C and D; error bars are SEM.