Human vocal fold fibroblasts isolation and culture
The research was submitted and approved by the ethics committee of XXXX (No. 2020014-1). All patients were informed in detail and signed consent forms to allow access to their clinic and ward information. Primary human vocal fold fibroblasts were isolated from surgical resected normal vocal fold of patients with hypopharyngeal carcinoma enrolled in XXXX.
Briefly, the tissues were lavaged in sterile distilled water three times, minced manually to fragments < 1mm3 and digested with 0.25% Trypsin-EDTA and Collagenase Type I for 1 h at 37°C. The digested solution was filtered through 70 µm cell strainer and centrifuged at 1000 rpm for 5 min. Precipitated cells were resuspended in high-glucose Dulbecco's modified eagle medium (DMEM, Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, USA) and 1% penicillin-streptomycin, seeded in the T75 cell culture flasks and maintained in the 37°C humidified incubator with 5% CO2.
Plasmid construction and cell transfection
The pWPI-LargeT-internal ribosome entry site (IRES)-green fluorescent protein (GFP) plasmids consisting of EF1α promoter and the Large T antigen were kindly shared by Dr. Pengyu Huang from Shanghai Tech University. 293FT cells were transfected with the pWPI-LargeT-IRES-GFP plasmids and packaging plasmids for recombinant lentivirus packaging. Viruses were collected, titred, and used for subsequent transfection on primary human vocal fold fibroblasts. The infection efficiency was the highest at an MOI of 100. Cell medium containing viruses was changed to fresh medium 72 hours after the transfection.
Immunofluorescence analysis
Primary or immortalized fibroblasts were seeded on glass slides. After incubation overnight at 37°C, cells were washed with PBS three times, fixed with 4% paraformaldehyde at room temperature for 30min, then permeabilized with 0.1% Triton X-100 for 1 h in the 37°C incubator. 10% FBS (Gibco, Cat# 10099141) was added to block the non-specific antigen. Primary antibodies applied in this assay include myofibroblast markers Vimentin (1:500, Abcam, Cat# ab92547, RRID: AB_10562134) and α-SMA (1:500, Abcam, Cat# ab7817, RRID: AB_262054). Goat polyclonal Secondary Antibody to Rabbit IgG (Alexa Fluor® 594, diluted 1:500) and Goat Anti-Mouse IgG2a (Alexa Fluor® 488, diluted 1:500) were chosen as secondary antibodies for 1h at room temperature. Counterstained with DAPI and sealed with glycerol, images were taken by a fluorescence microscope (Olympus, Tokyo, Japan).
Quantitative Real-Time Polymerase chain reaction analysis
Fibroblasts were lysed with TRIzol (Invitrogen, CA, USA) and total RNA was extracted. PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan) was applied to convert the total RNA into corresponding complementary DNA by reverse transcription. Real-time Polymerase chain reaction (RT-PCR) was performed using TB Green Premix ExTaq (TaKaRa, Tokyo, Japan) on the Applied Biosystems 7500 Fast Real-Time PCR System (BioRad, Richmond, CA, USA). The reaction system was comprised of 10 μL TB Green Premix Ex Taq (2x), 0.4 μL ROX Reference Dye II (50x), 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 2 μL cDNA template, and 6.8 μL sterile ddH2O. The PCR cycling program was 95°C for 30 s and the next 40 cycles of 95°C for 5 s, 60°C for 34 s. Melt curve at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The relative expression levels of target genes were normalized to GAPDH and analyzed by the comparative 2−ΔΔCT method. PCR samples were visualized by agarose gel electrophoresis. Primers used for PCR were listed as follows in Table 1.
Cell sorting
A Fluorescence Activated Cell Sorting (FACS) Ariall cell sorter (BD Biosciences) was used for cell sorting. Briefly, cell samples were resuspended into FACS buffer after trypsin digestion and phosphate buffered saline (PBS) washing. Single cell suspensions were prepared and the GFP positive cells were evaluated and sorted ultimately. Data were analyzed using FlowJo Software.
Western blot analysis
Western blot was applied to measure the protein content of Vimentin (54kDa), α-SMA (42kDa), FAP (95kDa) and fibronectin (262kDa). Total cellular proteins were extracted from cell lines and primary cells using RIPA lysis reagent (Biotechwell, Shanghai, China) containing the protease inhibitor phenylmethylsulfonyl fluoride (PMSF, Biotechwell, Shanghai, China). Protein concentrations were determined by the BCA kit (Beyotime, Shanghai, China). Samples were equally loaded in each lane (20 μg protein per lane), subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes (Biotechwell, Shanghai, China). Blocked with 5% non-fat milk in TBST at room temperature for 1 h, the membranes were then incubated with primary antibody at 4°C overnight. Primary antibodies were as follows: Vimentin (1:1000, Abcam, Cat# ab92547, RRID: AB_10562134), α-SMA (1:500, Abcam, Cat #ab7817, RRID: AB_262054), FAP (1:1000, Abcam, Cat #ab207178, RRID: AB_2864720), fibronectin (1:1000, Abcam, Cat #ab2413, RRID: AB_2262874), GAPDH (1:1000, Abcam, Cat #8245, 1:1000, RRID: AB_2107448). Next, the secondary antibody was added. The membranes were lavaged three times (10 min each time) with TBST and the bands were visualized with enhanced chemiluminescence (ECL) reagent.
Cell proliferation assay
Cell proliferative ability was quantified with the CCK-8 assays kit (Dojindo, Tokyo, Japan) according to the manufacturer’s instructions. In brief, fibroblasts were seeded in a 96-well plate at 2*103 cells per well. After attachment, at 0, 24, 48, 72, 96, 120 h, cells were treated with 10 μL of CCK-8 reagent and incubated for 1 h at 37°C. Absorbance was measured at a wavelength of 450 nm using Microplate Reader (Bio-Rad, Richmond, USA). Cells of each group were analyzed for 5 replicates.
Statistical analysis
Statistical analyses were performed with SPSS 26.0 (IBM, Armonk, NY) and Prism 9.0 software (GraphPad, San Diego, CA). Data were expressed as mean ± SD. Statistical significance was analyzed using Student’s t-test, Mann-Whitney-test, or one-way ANOVA tests as appropriate. P < .05 was defined as statistically significant. (* P < .05, ** P < .01, *** P < .001)