Primary culture of rabbit vibrissae DPCs and the establishment of immortalized cell lines
All work with rabbits and mice were approved by the Animal Care and Use Committee of Yangzhou University (Yangzhou, China, 14 March 2019, No. 201903005). Three four-month-old white rex-rabbits (female) were purchased from the Yuyao Xinnong Rabbit Industry. Rabbits were injected intravenously with zoletil-50 (6mg/kg) and waited to collect vibrissae skin after death. We tried to isolate DPCs three times in total. DPCs were isolated as previously described15. Cells were passaged in 0.25% trypsin and sub-cultured at a ratio of 1:3 when sub-confluent. DPCs are mesenchymal-derived follicular stem cells32,33，so we tried to culture them in MSCM (Mesenchymal Stem Cell Medium) complete medium (ScienCell, USA). primary DPC (passage 3) were transfected with pLVX-IRES-Puro-SV40LT lentiviral expression vector34 (Yingrun Biotechnology, Changsha, China). The puromycin resistance gene (Puro) was used for cell screening35. After 72 hours of transfection, DPCs were inoculated with MSCM containing 2.0 μg/mL puromycin for 7 days, and replaced with MSCM containing 1.0 μg/mL puromycin for 2 weeks. Cells were harvested in 0.25% trypsin, diluted with medium, and single cells were seeded into 96-well plates. Seven clones identified by immunofluorescence were selected and propagated. SV40T antigen expression was evaluated by immunofluorescence and RT-PCR analysis. Immortalized DPCs were cultured in MSCM medium. Immortalized DPC lines of passage 50 were used in this study.
Alkaline Phosphatase Activity
Primary DPCs (passage 2) and seven cell line(passage 10) were seeded into 24-well plates and cultured for incubated for 24 h. Cell alkaline phosphatase stain kits (Jiancheng Bioengineering Institute, Nanjing, China) were used to fix and stain the cells. Positive reactions were observed through gray-black particles or massive strip-like precipitates in the cytoplasm.
DPCs (passage 15) were seeded into 24 well plates and cultured for 24 hours. Cells were fixed in 4% paraformaldehyde for 30 min and washed with PBS. Cells were permeabilzed in 0.5% Triton X-100 for 1 hour, washed in PBS, and blocked for 30 min in 1% BSA (Boster, AR0004). Cells were then probed with primary antibodies (4 μg/mL) in blocking solution overnight. Primary antibodies against α-SMA (Boster, BM0002), VIM (Boster, BM0135), SV40LT (Santa Cruz (Pab 101): sc-147) were used. Cells were then washed and labeled with secondary fluorochrome-conjugated antibodies (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488), abcam, ab150077, Cy3–conjugated Affinipure Goat Anti-Mouse IgG(H+L), proteintech, SA00009-1) for 2 hours at room temperature (25˚C). Finally, cells were stained with DAPI (Beyotime, C1006) for 10 min and imaged under a fluorescence microscope.
Total RNA was isolated using the RNA simple Total RNA Kit (Tiangen Biotech, Beijing, China) and used as a template for cDNA synthesis with HiScript Q Select RT Super Mix for qPCR Kit (Vazyme Biotech, Nanjing, China). The synthesized cDNA was used as a template for gene amplification using primers and 2×Rapid Taq Master Mix(Vazyme Biotech, Nanjing, China). Primer sequences are summarized in Table S1. PCR conditions are summarized in Table S2-S3. PCR products were separated by 1% agarose gel electrophoresis and observed on a gel doc ez imager (BIO-Rad, shanghai, China).
Soft agar assays
Pre-heated 1.2% thawed agar was mixed with DMEM medium containing 20% fetal bovine serum as the basal layer36. B16F10 and DPCs (1 × 103 cells/mL)were mixed with the pre-heated 0.7% agarose ensuring no air bubbles were generated. Cells were spread onto agarose plates and 200 ul of complete medium was added every two days. Colony formation was observed via microscopy for up to 2 weeks.
1.5μl colchicine (1 mg/ml) to was added to DPCs in 25 cm2 cell culture flasks for 4 h at 37˚C, 5% CO2. Cells were collected and low-permeated in 0.075 M KCl at 37 ˚C for 32 min. Cells were collected, fixed in 3:1 methanol: acetic acid three times for 30 min, 15 min and 15 min. Finally, cell suspensions were dripped onto pre-cooled slides and stained with working fluid prepared by Giemsa (Amresco, Solon, OH, USA).
Four-week-old female BALB/c nude mice were purchased from the College of veterinary medicine (Yangzhou University, China) (http://syxy.yzu.edu.cn/art/2017/6/6/art_40971_559648.html). Cells (5x106) were resuspended in 200 μl PBS. Subcutaneous injection with PBS was performed into the right armpit of mice (n=33)29. Mice were observed weekly for 3 weeks and sacrificed with carbon dioxide. Mouse melanoma cells (B16F10) were used as a positive control.
Cell cycle analysis
To analyze cell cycle progression and apoptosis in primary DPCs (passage 2) and R-7 (passage 50), the Cell Cycle and Apoptosis Analysis Kit (Beyotime, Jiangsu, China) was used. Collected cells were incubated overnight in 70% ethanol and treated for 5 min with RNase A. After 30 min of incubation with propidium iodide (PI) in the dark at 37˚C, DNA content was analyzed on a Flow Cytometer through fluorescent analysis (LSRFortessa, BD Company, American).
Cell growth curves
Primary DPC (passage 2), R-7 (passage 5, passage 19 and passage 50) were harvested and seeded into 24-well plates at a density of ~103 cells/mL (n=3 per passage). Then Cells were counted every 24 h using an Automatic cell counter (BIO-Rad, shanghai, China) continuously for 8 days. Uncounted cells were replaced with fresh MSCM medium every 2 days.
Cell proliferation assessments
Cell Counting Kit-8 (CCK-8) (Vazayme, Nanjing, China) assays were used to detect cell apoptosis. Cells (105 per well) were seeded into 96-well plates and cell viability assessments were performed through the addition of CCK-8 (10μL) for 2-4 hours. The optical density of each well was determined at 0, 24, 48, and 72 hours at 450 nm with Infinite M200 Pro (Tecan, Männedorf, Switzerland).
Cell lysates were obtained using RIPA Lysis Buffer (PPLYGEN, Beijing, China). Protein concentrations were determined with the Enhanced BCA Protein Kit (Beyotime)37. These proteins were detected and analyzed using the Wes automated Western Blot Analysis System38. The following antibodies were used: 1:200 Anti‐SV40LT (Santa Cruz (Pab 101): sc-147), 1:200 Anti‐GAPDH mouse monoclonal antibody (Abcam, ab8245), 1:100 Anti‐Vimentin (VIM) monoclonal antibody (Boster, BM0135), 1:100 Anti-α smooth muscle actin(α-SMA) antibody monoclonal antibody (Boster, BM0002). Expected protein sizes, SV40LT:92 kDa, VIM:55 kDa, α-SMA:43 kDa, GAPDH: 39 kDa.
SPSS 22.0 was used for data analysis. Each analysis has three biological replicates, and all error bars in the results represent the mean ± SD.*p <0.05 were considered significantly different, and **p<0.01 considered extremely significantly different.