2.1 Human tissue samples
HHD skin samples were obtained from patients (n = 22) with HHD by examining their clinical features and histopathology. Normal skin tissues were collected from healthy donors (n = 20) undergoing plastic surgery. For cell experiments, human foreskin tissues were isolated from healthy patients who underwent circumcision. This study was performed under the guidance and supervision of the Hospital Research Ethics Committee, and written informed consent was obtained from all subjects.
2.2 Immunohistochemistry
As described previously [9], tissues were cut into 5-µm paraffin sections. The sections were subjected to dewaxing treatment, followed by antigen retrieval with sodium citrate. After sealing with 2% goat serum (Beyotime Biotech, Shanghai, China), the samples were incubated with primary antibodies at 4℃ overnight. These primary antibodies were mouse antibodies against SPCA21 (Proteintech, Rosemont, Illinois, USA) and F-actin (Abcam, Cambridge, MA, USA) and rabbit antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Proteintech), occludin (Abcam), claudin1 (Abcam), claudin4 (Abcam), ZO-1 (Abcam), and P-cofilin (Cell Signaling Technology, Danvers, MA, USA). Then the sections were incubated with a horseradish peroxidase-labeled goat anti-rabbit antibody (DAKO, Glostrup, Denmark) and the 3,30-diaminobenzinechromogen substrate (DAKO). Phosphate-buffered saline controls were used for all samples.
2.3 Cell culture and short hairpin RNA (shRNA) transfection
Keratinocytes were obtained as described previously [4], and cultured in the keratinocyte serum-free medium (KSFM; Sciencell, San Diego, CA, USA). The cultured cells at passage 2 or 3 were divided into blank, control shRNA, and ATP2C1 shRNA lentivirus transfection groups and seeded on 6-well plates. A total of 20 µL of shRNA lentivirus and enhancement solutions (Genechem, Shanghai, China) were added to the culture medium of the ATP2C1 shRNA group. The same amounts of the negative virus and enhancement solution were added to the control shRNA group. After culturing for 72 h, the keratinocytes were further cultured in the KSFM containing 1 µg/mL puromycin. When the blank-group cells were completely killed by puromycin and no more cells died in the ATP2C1 shRNA group, the surviving cells were used for further experiments.
2.4 Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
Total RNA was extracted from fresh tissues and cell cultures using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed into complementary DNA (cDNA) using a commercial cDNA kit (Takara Bio, Shiga, Japan). Then, qPCR was performed on 7900HT Fast PCR System (Applied Biosystems, Waltham, MA, USA). SYBR Green Master Mix (Takara Bio, Shiga, Japan) was used as a fluorescent dye. The synthesis and detection of all target gene primers were performed by Bioscience (Shanghai, China) using GAPDH primers as internal references. The primer sequences are listed in Table 1.
2.5 Cellular immunofluorescence
The cells were cultured in glass-bottom culture dishes (MatTek, Ashland, MA, USA) and fixed with the 4% paraformaldehyde solution. The cells were permeabilized using Triton and subsequently blocked using a goat serum sealer. Then, the cells were incubated with primary antibodies (see above under ‘‘Immunohistochemistry’’) at 4°C overnight. Subsequently, fluorescence secondary antibodies and TRITC-labeled rhodamine-conjugated phalloidin (Beyotime, Shanghai, China) were sequentially added for staining. After incubating with the 4',6-diamidino-2-phenylindole solution (Het Biotech, Xi'an, China), the cells were observed under a digital confocal microscope (Leica, Wetzlar, Germany).
2.6 Western blotting
Protein lysates were prepared from the cell cultures using the radioimmunoprecipitation buffer (Het Biotech). Protein concentrations were determined using Bicinchoninic Acid Protein Assay Kit (Het Biotech). The samples were separated on electrophoresis gels, followed by transfer onto polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). Horseradish peroxidase goat antirabbit immunoglobulin G (DAKO) was used as a secondary antibody. The protein lysates were subjected to chemiluminescent detection using an electrochemiluminescence kit (Millipore). The obtained band intensities were quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
2.7 Transmission electron microscopy
The cells were fixed using the electron-microscopy-specific fixative, followed by neutral phosphate buffer-osmic acid addition. After dehydration, infiltration, and embedding, the cells were cut into 60–80 nm ultra-thin sections. Following double staining with 1% uranyl acetate and lead citrate, the sections were visualized and images were captured with a transmission electron microscope(HT7700, HITACHI, Tokyo, Japan).
2.8 Cell proliferation and apoptosis
The cells were cultured in 96-well plates and transfected with shRNA. After transfecting for 24, 48, and 72 h, the cultures were analyzed for cell proliferation. The cell counting kit-8 (CCK-8) solution (Beyotime) was added and the cells were incubated at 37°C for 2 h. Absorbance was measured at 450nm.
Cell apoptosis was determined by flow cytometry. The cells were diluted to 1 × 106 cells/mL, incubated with a binding buffer, and mixed with 7-aminoactinomycin D (red fluorescence) and annexin V-phycoerythrin (orange-red fluorescence). The data were analyzed using the LSRII instrument (BD Biosciences, San Jose, CA, USA) and subsequently processed using the FlowJo7.6.1 software (Tree Star, Ashland, OR, USA).
2.9 Intracellular Ca2 + detection
Cells in the three groups were transferred to 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. The culture medium was then replaced with a medium containing fluo-3 AM (Beyotime) and the cells were further incubated at 37°C for 60 min for fluorescence probe loading. After the conversion of fluo-3 AM into fluo-3, fluorescence microplate detection was performed to record and export the data.
2.10 Statistical analysis
The experimental results were subjected to statistical quantitative analysis and graphing using the ImageJ and GraphPad Prism 8.0 software. A two-sample independent t-test was performed for comparing any two groups, and analysis of variance was performed for comparing the three groups. Differences were considered statistically significant at p < 0.05.