Itga6

Cells were fixed with 4% cold paraformaldehyde for 10 minutes, permeabilized with 0.2% Triton X-100 for 10 minutes at room temperature and blocked with 10% horse serum (Vector Laboratories) in 0.2% Triton X-100 for 1 hour at room temperature. Cells were incubated, shaking, overnight at 4°C with primary antibodies. Primary antibodies used in this study are: Gibbin/AHDC1 (1:25, Abcam), KRT14 (1:400, BioLegend), ITGA6 (1:200, Millipore, MAB1378), IVL (1:100, Abcam), DSG3 (1:100, Thermo Fishers), KRT10 (1:500, Covance), COL7A1 (1:250, Millipore), HP1a (1:1000 Santa Cruz), and p63 (1:100 GeneTex). Cells were then incubated with Alexa 488, 555, or 647-conjugated secondary antibodies diluted with 0.2% Triton X-100 in PBS (1:500, Life Technologies) for 1.5 hours at room temperature. Slides were washed three times in PBS, incubated with Hoescht (1:10,000, Life Technologies) for 15 minutes, and mounted onto slides with Prolong Gold (Life Technologies). All fluorescence images were taken using an SP5 confocal laser scanning microscope (Leica) using the Leica Application Suite for Advanced Fluorescence software version 2.7.9. Live cell images were taken with a Leica Live Cell Camera. All images are representative of 10-20 random views of each sample. HP1α foci were quantified and analyzed with a custom python script.

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 30 min, and blocked with 10% horse serum (Vector Laboratories) in 0.2% Triton ×−100 in PBS for 30 min at room temperature. Cells were incubated overnight at 4C with primary antibodies. Primary antibodies are: K18 (1:800, R&D AF7619), K14 (1:800, BioLegend SIG-3476–100); AP-2γ (1:100, Cell Signaling 2320), p63 (1:100 Gene Tex GTX102425), ITGA6 (1:200, Millipore, MAB1378). Then the cells were incubated with Alexa 488, 555 and 647-conjugated secondary antibodies diluted with 0.2% Triton X-100 in PBS (1:500, Life Technologies) for 1 hour at room temperature. Following three washes with PBS, slides were mounted with the Prolong Gold mounting medium (Life Technologies). Prior to mounting, slides were incubated with 1:10000 Hoechst for 10 min. The fluorescence images were taken using the TCS SP2 confocal laser scanning microscope (Leica). In this assay, the cell culture was performed over three times and subsequent cell staining was done by different investigators. For each slides with same antibodies staining, the images were randomly taken from 5–10 individual regions using same parameters and the representative one was shown. Slides stained only with secondary antibodies were used as a control to determine the false positive signal.