Establishment and characterization of immortalized myoepithelial cells of sweat glands
Human skin tissue
Human skin tissue was obtained with the informed consent of the Kinugasa Clinic (Osaka, Japan) and Bizcom Japan (Tokyo, Japan). The experiments using human skin have been approved by the ethics committee of Osaka University. All research was carried out in accordance with the guidelines and regulations in force. Informed consent was obtained from all participants.
Isolation of sweat glands and culture of spheroid cells
Sweat glands were obtained by taking skin biopsies using tweezers and scissors and disaggregated with 600 U / mL type II collagenase (Worthington Biochemical Corporation, Lakewood, NJ) in Mammocult Human Medium ( Stemcell Technologies, Vancouver, BC, Canada) for 4 to 16 hours at 37 Â° C and 5% CO2 using a tube rotator (NISSIN, Tokyo, Japan) at 20 rpm. After the enzymatic reaction, the sweat glands were isolated using micropipettes (P1000). The isolated sweat glands were then incubated sequentially with 0.5% trypsin-EDTA (Gibco, Waltham, MA, USA) and 5 U / mL dispase (Gibco) at 37 Â° C by pipetting to dissociate cells from the sweat glands. . The cells were then filtered through a 40 Î¼m filter (Falcon, Corning, NY), centrifuged at 350xg for 5 min at 4 Â° C, and washed three times with Dulbecco’s phosphate buffered saline (Gibco). Washed cells were seeded in 24-well ultra-low attachment culture plates (Corning Inc., Corning, NY, USA) with spheroid culture medium (Supplementary Table S1) at a density of 2500 cells / well. The sweat gland spheroids were enzymatically dissociated into single cells and re-cultured under the same conditions. The spheroids were passed once every seven days.
Cells were fixed with 4% paraformaldehyde for 15 min at 25 Â° C. Fixed cells were mounted on slides and incubated at 37 Â° C for 8-16 hrs for cell attachment to the slides. Cells attached to the slides were permeabilized with 0.5% Triton X-100. The cells were then blocked with 3% bovine serum albumin (BSA) solution for 30 min and incubated with the primary antibody diluted in 3% BSA solution overnight (16 h) at 4 Â° vs. Subsequently, cells were washed three times with 0.05% Triton X-100 in phosphate buffered saline (PBS-T) and incubated with the secondary antibody diluted in 1% BSA solution. for 1.5 h at 25 Â° C. After incubation with the secondary antibody, cells were washed three times with PBS-T and stained with 4 â², 6-diamidino-2-phenylindole (DAPI) for 5 min at 25 Â° C. After washing the slides with PBS, the cells were covered with glass slides. Fluorescence images were obtained using a BZ-X710 microscope (Keyence Japan, Osaka, Japan) at 20X magnification. The antibodies used are listed in Supplementary Table S2.
Assay of -galactosidase (Î²-gal) activity associated with senescence
The cells fixed on slides were stained using SPiDER-Î²gal (Dojindo, Kumamoto, Japan), according to the manufacturer’s protocol. Then the cells were washed three times with PBS and stained with DAPI for 5 minutes at 25 Â° C. After washing the slides with PBS, the cells were covered with glass slides. Fluorescence images were obtained using a BZ-X710 microscope (Keyence Japan, Osaka, Japan) at Ã 20 magnification.
Lentivirus vectors carrying the hTERT and SV40Tt genes were purchased from Applied Biological Materials (Richmond, BC, Canada). Spheroid-forming sweat gland cells were immortalized by infection with viral medium (spheroid culture medium without Matrigel (BD Biosciences, San Jose, CA) and antibiotic-antimycotic (Gibco), 5 Ã 106 U / mL of lentivirus vectors and 10 g / mL of polybrene (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The viral medium was replaced with fresh medium after 6 h.
Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted using the TRI reagent (MRC, Cincinnati, OH) according to the manufacturer’s instructions, and complementary DNA (cDNA) was synthesized from the RNA using the kit. reverse transcription method QuantiTect (QIAGEN, Hilden, Germany). The synthesized cDNA was subjected to real-time PCR analysis using the THUNDERBIRD SYBR qPCR mix (TOYOBO, Osaka, Japan) and a ViiA7 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primer sequences used for PCR are listed in Supplementary Table S3.
Agarose gel electrophoresis of PCR products
CDNA obtained from total RNA was amplified by PCR using Veriti Thermal Cycler (Applied Biosystems). The amplified cDNA was subjected to 2% agarose gel electrophoresis for 30 minutes using Midori Green (Nippon Genetics, Tokyo, Japan). DNA band images were obtained using Limited-STAGE (AMZ Systems Science, Osaka, Japan). Targeted bands were analyzed using ImageJ software version 1.53 (NIH, Bethesda, MD, USA, http://imagej.nih.gov/ij/).
Culture of immortalized myoepithelial cells with small compounds
Immortalized myoepithelial cells were seeded in 24-well ultra-low attachment culture plates (Corning) containing spheroid culture medium (10,000 cells / well). The cells were cultured with each compound from the library consisting of 99 small compounds, i.e. targeting factors related to the maintenance and differentiation of stem cells (Selleck Chemicals, Houston, TX, USA) ( supplementary table S4) for seven days. Expression of K18 in cultured cells was measured by RT-PCR.
Western blot analysis
Cells were lysed in solution (50 mM Tris-HCl [pH 7.5], 125 mM NaCl, 1.0% Nonidet P-40) and subjected to Western blot analysis with 12% polyacrylamide gel and polyvinylidene fluoride membrane. The antibodies used in the western blot are listed in the supplementary table S2. Target proteins were detected using ECL prime solution (GE Healthcare, Chicago, IL, USA) and Amersham Imager 600 (GE Healthcare). Targeted bands were analyzed using ImageJ software version 1.53 (NIH, Bethesda, MD, USA).
All data is represented by the mean Â± standard error of the mean. Statistical significance was determined by unpaired two-tailed Student’s t-test, Mann-Whitney U test, Steel-Dwass test, and Tukey-Kramer test using Prism 7 version 7.04 software (GraphPad Software, San Diego, CA, https://www.graphpad.com/scientific-software/prism/). The differences were considered statistically significant at P