Involucrin

Cells for analysis were lysed in 2 × SDS-PAGE lysis buffer and proteins were separated by SDS-polyacrylamide gel electrophoresis. After electro-transfer in Towbin buffer (2.5 mM Tris/19.2 mM Glycine/0.1% SDS/20% methanol), membranes were blocked in 5% non-fat milk protein in TBS/0.1% Tween (TBS-T). Membranes were then incubated at 4 °C overnight with primary antibody to involucrin (Sigma-Aldrich, Gillingham, UK), VDR (Cell Signaling Technology, Leiden, The Netherlands), β-Actin (Abcam, Cambridge, UK) or GAPDH (Abcam) in 5% non-fat milk protein/TBS-T; or to Brk (ICR-100), in 5% BSA/TBS-T. Proteins were visualised with an appropriate hrp-conjugated secondary antibody (Dako, via Agilent, Stockport, UK) and chemiluminescent substrate (Pierce, via Thermofisher, Loughborough, UK).

Tissue sections were either stained with hematoxylin–eosin (H&E) or exposed to Tris EDTA [pH 8.0] for heat-induced antigen unmasking and subsequent incubation for 40 min with the appropriate primary antibody to 5.8S rRNA (1:1500 dilution, Santa Cruz, cat. #sc-33678), involucrin (1:100 dilution, Sigma-Aldrich, cat. #I9018) or keratin 14 (1:300 dilution, Biolegend, cat. #905301). Immunohistochemical staining was carried out using a Ventana Discovery Ultra instrument (Roche, cat. #05987750001). For standard staining, the Discovery OmniMap anti-rabbit horse radish peroxidase detection system (Roche, cat. #760-4311) was used for detection as specified by the manufacturer. For immunofluorescent studies, sections were incubated for 1 h with appropriate secondary antibodies to either rabbit or mouse IgGs labeled with Alexa Fluor 488 (1:200 dilution, ThermoFisher, cat. #A21206) and Cy3 (1:200 dilution, Jackson ImmunoResearch, cat. #115-165-146). For staining of nuclei, sections were incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, cat. #D9542) for 5 min. Immunohistochemical signals were quantified using Fiji software.

Immunofluorescence staining was performed on frozen graft tissue sections after 10-min fixation with ice-cold acetone and/or methanol (7 μm thickness). Sections were blocked for 1 hour at room temperature (RT) with 3% fetal bovine serum in phosphate buffered saline before incubation with primary antibodies against hC7 LH7.2 (Sigma-Aldrich) in a 1:500 dilution (Supplementary Table S1 online), desmoglein-1 (Fitzerald Industries, Acton, MA), involucrin (Sigma-Aldrich), keratin 10 (in-house), complex IV subunit II MTCO2 (Abcam, Cambridge, UK) overnight at 4 °C. Secondary antibody incubation with Alexa Fluor goat antimouse 488 (Invitrogen, Paisley, UK), goat antirabbit Cy3 (Life Technologies, Paisley, UK), and strep 488 was followed for 1 hour at RT. Sections were stained with 4'.6-diamidino-2-phenylindole (5 mg/ml) and mounted using a ProLong Gold antifade agent (Life Technologies). These were also stained by a hematoxylin and eosin histochemical technique. Staining was visualized and imaged using a Leica DMLB upright microscope (Leica Microsystems CMS, Wetzlar, Germany) and a Zeiss Axiophot 2 (Zeiss, Oberkochen, Germany) and processed using Image-Pro 6.2 (MediaCybernetics, Rockville, MD). Confocal imaging was carried out on a Zeiss LSM 510 Meta laser confocal microscope (Zeiss). Postprocessing was carried out using ImageJ.

Immunostaining was performed for the expression of keratinocytes specific markers: CK5, CK10, involucrin (Sigma Aldrich, USA), and loricrin (Abcam, UK) and dermal fibroblast specific markers, collagen-3 (Santa Cruz Biotechnology, USA), desmin (Santa Cruz Biotechnology, USA), FAP-α (Abcam, UK), and procollagen-1 (Santa Cruz Biotechnology, USA). Briefly, cells were washed with PBS three times and treated with 4% paraformaldehyde (PFA) for 15 minutes. The fixed cells were washed with PBS (5 × 3 times) and incubated with the primary antibodies overnight at 4°C. Incubation at room temperature with respective secondary antibodies (1 : 200) was performed for 1 hour at 37°C. After washing with PBS, cell nuclei were stained with DAPI (Sigma Aldrich, USA) and observed microscopically.

Cells were lysed with 20 mM Tris pH 7.5, 150 mM NaCl, 0.5% Deoxycholic acid, 10mM EDTA and 0.5% Triton X‐100 containing protease inhibitor cocktail (complete ULTRA tablets, Roche). Histones were extracted from cells by acid extraction method (Halsall et al, 2015). According to standard procedure, lysates were treated with loading buffer, separated by 12–10% SDS–PAGE, transferred onto nitrocellulose membrane (Bio‐Rad) using Trans‐Blot Turbo Transfer System (Bio‐Rad) and immunoblotted with following primary antibodies: JARID2 (1:1,000, Cell Signaling Technology, USA; 1:1,000, GTX129020, GeneTex), EZH2 (1:1,000, Cell Signaling Technology, USA), involucrin (1:1,000, Sigma), transglutaminase‐1 TGase‐1 (1:1,000, Santa Cruz Biotechnology, INC), c‐Jun (1:1,000, Cell Signaling Technology, USA) and H3K27me3 (1:1,000, 07‐449 Millipore). GAPDH (1:1,000, ThermoFisher Scientific) and C‐terminus of histone H3 (1:5,000, Abcam) are used as loading controls. Immunoblots were visualised using fluorescence detection and scanned using odyssey infrared detection system (LI‐COR Biosciences). Densitometry analysis was done using Image Studio Lite (LI‐COR Biosciences).

Freshly isolated keratinocyte subpopulations were fixed in 4% buffered PFA and cytospun onto glass slides. Cells were permeabilized with Triton × 100 0.1%, stained for K10 (clone EP1607Y, Epitomics, Burlingame, CA, USA), K15 (EPR1614Y, Epitomics), and Involucrin (clone I9018, Sigma) and then labeled with Alexa Fluor secondary antibodies, Alexa Fluor 546 and 488-conjugated goat IgGs (Invitrogen, Waltham, MA, USA). Then slides were stained with 1 μg/mL Dapi (Sigma). Immunofluorescence on skin equivalent slides was performed as in IHC methods except for the different primary antibodies: K10, K15, and Survivin (Novus Biologicals, Littleton, CO, USA) and the secondary antibodies Alexa Fluor 546 and 488-conjugated goat IgGs (Invitrogen). Micrographs were taken on a Confocal Scanning Laser Microscopy (Leica TCS4D) (Leica, Exton, PA, USA). Quantification of immunofluorescence staining was performed by analyzing 6 representative fields for each staining sample, using ImageJ software. Scoring was made by means of positive cell counting.