Generation and characterization of Myo5B KI cells. (A) Scheme of ZFN-mediated double-strand break and homologous recombination via a short oligonucleotide to introduce the 1125G>A premature stop codon mutation into the CaCo2 genome. (B) Western blot showing Myo5B protein levels of WT, Myo5B KI, and HS-Myo5B–reverted cells. (C) Cytoplasmic mRNA expression levels of Myo5A and Myo5C are not altered upon the depletion of Myo5B. Expression levels were assessed by qRT-PCR. Three independent experiments. (D) Polarity is disrupted upon Myo5B depletion in 3D cyst formation assay in comparison to WT cells (left to right: 24 h, 48 h, 3 d, and 7 d). Bars, 10 µm. (E) Quantification of single lumen formation. n ≥ 100 cysts/experiment; three independent experiments. (F) Scanning electron micrographs of the apical brush border in 2D CaCo2 cultures. Microvilli of Myo5B KI cells are reduced in size and number. Bars, 5 µm. (G) Transmission electron micrographs of the apical part of CaCo2 show scarce, short microvilli (arrows) and prominent subapical vesicle accumulations (arrowheads) in Myo5B KI cells. Bars, 1 µm. Mean ± SD. ***, P < 0.005. n.s., not significant.