Fig 2: (A) Shear test measuring cell sheet cohesion shows significant diminishment in cohesion strength with disruption of each component of the cytoskeleton using toxins, with acrylamide treatment exhibiting the greatest decrease in cell sheet cohesion. *p<0.05 compared to the matched untreated control. Unnormalized times in seconds: con/CytD=140/73; con/noco=93/40; con/acryl=148/45. (B) Shear test shows double siRNA knockdown of keratin 5 and keratin 14 weakens cell sheet cohesion. *p<0.05 and **p<0.01. Unnormalized times in seconds: control=145; negative control=150; K5=113; K14=98; K5+K14=45. (C) Immuno-staining and (D) western blot analysis of siRNA transfection on adhered cells demonstrates reduction of K5 or K14 expression levels. K5/K14 knockdown together significantly reduced both K5 and K14 expression levels. Scale bar is 10 µm. GAPDH was used as a loading control.

Fig 3: Effect of osmotic shock on the keratin cytoskeleton in the two cell lines studied.A. NEB-1 K14wt-GFP (upper panel) and NEB-1 K14R125P-GFP (lower panel). Both cell lines were subjected to hypo-osmotic shock. Clear peripheral aggregates were seen in R125P cells 20 and 30 min after osmotic shock. No filament fragmentation or aggregates were observed for NEB-1 K14wt-GFP cells. Scale bar = 15 µm. B. Insets from Figure 3A, 20 min after osmotic shock, showing K14 aggregates in the NEB-1 K14R125P-GFP cells, and reconstituted K14 filaments in the NEB-1 K14wt-GFP cells.

Fig 4: Basal/myoepithelial differentiation of DKAT xenografts. A.DKAT xenograft tumor stained with DAPI (blue) nuclear counter stain, CK14 (green) showing basal/myoepithelial differentiation of tumor cells, and human-specific p53 (red) confirming that both the CK14-positive cells and the more luminal, CK14 weak/negative cells are human DKAT tumor cells. 100 micron scale bar in the DAPI channel applies to all four images. B. DKAT tumor from a second mouse at higher magnification shows an area of vague glandular architecture with somewhat elongated CK14-positive basal/myoepithelial cells (arrows) surrounding CK14 weak/negative cells which surround a poorly formed glandular lumen (asterix). 25 micron scale bar show in in DAPI channel.

Fig 5: Analysis of HDR Efficiency(A) PCR analysis of genomic DNA from CRISPR/Cas9-treated EBS hKc using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14. To minimize the size of the PCR product and to facilitate subcloning, we performed a nested PCR using a forward primer binding to exon 6 and a reverse primer binding to intron 7, resulting in a specific product of 507 bp. An EcoRI restriction digest confirmed the presence of recombined KRT14 alleles in treated EBS cells. Upon bacterial transformation, single colonies were picked for colony PCR and amplified product digested with EcoRI to identify recombined alleles. (B) Sequence analysis of one representative single clone, containing a modified wild-type allele (green arrow: wild-type sequence at mutation site [position 178] within exon 6), is shown. The allele additionally contains the introduced restriction sites NheI/EcoRI and the silent mutations (blue stars). Sequence alignment was performed with the software “Multiple sequence alignment with hierarchical clustering.”21