CRISPRi-iAT2s were transduced with DSP gRNA and treated without (−dox) or with (+dox) doxycycline. (A) Immunofluorescence staining of desmoplakin (DSP). DSP, red; nuclei, blue; scale bar, 10 μm. (B) Flow cytometry analysis of DSP expression, represented by mean fluorescence intensity (MFI). n = 3 experimental replicates of independent wells of a differentiation; error bars represent SD. Statistical significance was determined by unpaired, two-tailed Student’s t test; ***P < 0.001. (C) Transmission electron microscopy images showing tight junctions (TJ) and desmosomes (De); scale bar, 200 nm. (D) Violin plots showing module scores for KEGG (Kyoto Encyclopedia of Genes and Genomes) tight junction, cell adhesion molecules, and gap junction. (E) Dot plots showing expression of genes associated with desmosome, (F) adherens junction, and (G) TJ. Genes that were not expressed by iAT2s were excluded from dot plots. (H) Immunofluorescence staining of claudin-4, ZO-1, and E-cadherin. (I) Immunofluorescence staining of keratin-18 and α-tubulin. Arrows indicate disorganized filaments or tubules. Protein of interest, red; nuclei, blue; scale bar, 10 μm.