Materials and reagents
Antibodies specific for LCK, SOCS1 were purchased from Abcam (Abcam, Cambridge, UK). The antibodies for α-SMA, Col1α1, JAK1, p-JAK1, STAT1, Pp-STAT1, STAT3, p-STAT3 and β-actin were purchased from Bioss Biotechnology (Bioss, Beijing, China). An alanine aminotransferase (ALT) (C009-2-1) assay kit and aspartate aminotransferase (AST) (C010-2-1) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TGF-β1 was purchased from Peprotech (New Jersey, USA). An Annexin V-FITC Cell cycle analysis kit (BB-4104), and Cell Counting Kit-8 (CCK-8) (BB-4202) were obtained from BestBio (Shanghai, China). Mouse Hyaluronic Acid (HA), laminin (LN) and Type 3 procollagen (PCIII) ELISA kits were obtained from jymbio (Jiyinmei Biotechnology, Wuhan, China).
Mouse models of CCl4-induced liver fibrosis
Male C57BL/6J mice (six- to eight-week-old) were obtained from the Experimental Animal Center of Anhui Medical University. All animal procedures were approved by the Institutional Animal Experimental Ethics Committee. Mice were randomly allocated to each group, food and water were freely available throughout the experiments and maintained in a 12-h light/12-h dark cycle. Liver fibrosis was established by intraperitoneal injection of CCl4 (0.001 ml/g, CCl4 dissolved in olive oil at a ratio of 1: 4), biweekly for 4 weeks. Control mice were injected with the same volume of olive oil. The reversal model was kept normal feeding for 6 weeks after CCl4 stop. Then, serum samples and liver tissues were collected for further analysis.
Adeno-Associated Virus 9 Mouse Model
Luciferase-labelled specific liver tissue location of rAAV9-shLck and vector were designed and obtained from Hanheng (Shanghai, China). All mice were randomly divided into 5 groups (n = 6 per group): vehicle group, CCl4 group, rAAV9-NC group, rAAV9-NC + CCl4 group, and rAAV9-shLCK + CCl4 group. Two weeks later, mouse LF model was established for 4 weeks after rAAV9 administration. Mice exposed to rAAV9 delivery were anaesthetized, effect of rAAV9-shLCK on liver tissue location was confirmed using an IVIS Lumina III Imaging System (Caliper Life Sciences, USA). Vehicle group mice were administered intraperitoneal injection olive oil biweekly. CCl4 group was induced by CCl4 intraperitoneal injections (0.001ml/g) biweekly. rAAV9-NC group mice were injected with an empty rAAV9 vector by tail vein injections and intraperitoneal injection olive oil. rAAV9-shLCK group mice were injected with an rAAV9-shLCK vector by tail vein injections and intraperitoneal injection olive oil. AAV9-NC + CCl4 group mice were injected with an empty rAAV9 vector by tail vein injections and CCl4 intraperitoneal injections (0.001ml/g) biweekly. rAAV9-shLCK + CCl4 group mice were injected with an rAAV9-shLCK vector by tail vein injections and CCl4 intraperitoneal injections (0.001ml/g) biweekly. Then, collected serum samples and liver tissues which were paraformaldehyde-fixed and paraffin-embedded.
Cell culture and cell treatment with TGF-β1
HSC-T6 cells, the rat HSCs, were obtained from Procell Life Science & Technology (Wuhan, China). HSC-T6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone, USA), supplemented with 10% fetal bovine serum (FBS, Every Green, China) and incubated at 37°C with 5% CO2. HSC-T6 cells were induced by 10 ng/ml TGF-β1 for 48 h.
In vitro activation of HSC-T6 was achieved by co-culturing HSC-T6 cells with TGF-β1 (10 ng/ml) for 24 h. After that, they were treated with the adipogenic differentiation mixture (MDI, 0.5 mM isobutylmethylxanthine (Sigma-Aldrich, St. Louis, MO, USA), 1 µM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), and 1 µM insulin (Sigma-Aldrich, St. Louis, MO, USA)) and incubated for 48 h.
Histopathology and immunohistochemistry staining
Paraformaldehyde-fixed, paraffin-embedded liver tissues were sectioned (5 µm) for H&E and Masson and immunohistochemical (IHC) staining for α-SMA and Col1α1. Slides were scanned by an automatic digital slide scanner (Pannoramic MIDI, 3DHISTECH, Hungary) and analysed by the CaseViewer software. The positive staining areas were measured by Ipwin32 software.
To discover LCK and SOCS1 location in liver tissues, frozen liver tissues of mice were blocked with 10% BSA at 37°C for 30 min to avoid nonspecific staining. Then, the sections were incubated with mixture of rabbit polyclonal primary antibodies for LCK (1:100) or SOCS1(1:100) and mouse polyclonal primary antibodies for α-SMA (1:400) at 4°C overnight. Sections were then incubated with a secondary conjugated antibody (1:100) in the dark at room temperature for 1 h. The nuclear staining was achieved by dropping DAPI for 5min. The stained sections were examined by inversion fluorescence microscopy until tissue section were dry.
To further discover the expression of LCK in non-treated HSCs, TGF-β1-treated HSCs, MDI-treated HSCs in vitro, we performed cell immunofluorescence mono-staining. Three cells groups were washed, fixed, permeabilized and blocked with 5% bovine serum albumin (BSA). The cells were incubated with a rabbit monoclonal primary antibody for LCK (1:100) at 4°C overnight. The second day, anti-rabbit FITC conjugated secondary antibodies was used to conjugate antigen combined LCK for 1h at room temperature. The cells were counterstained with DAPI and visualized using fluorescence microscopy. Cell nucleus was shown as blue fluorescence and LCK was green fluorescence.
HSC-T6 cells were seeded into 96-well plates, and the edge wells were filled with PBS. Cells were treated with 10ng/ml TGF-β1 or MDI after transfection with siRNA and pEX-2-LCK. After 24h or 48h,10 µl CCK-8 (Shanghai, China) was added to each well for 4 h. The value of absorbance (A) was examined at a wavelength of 450 nm. The cell viability was calculated according to the formula.
Mice liver tissues (30 or 50 mg) and HSC-T6 cells were lysed with RIPA lysis buffer (Beyotime, China). The protein concentration was determined by using a BCA protein assay kit (Beyotime, Jiangsu, China), according to the instruction manual. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp, Billerica, MA, USA). The PVDF membranes were blocked in 5% skim milk for 1.5 h at room temperature and then, washed three times in TBST. The PVDF membranes were incubated at 4°C overnight with specific primary antibodies which were diluted in Dilution Buffer (Beyotime, China). Rabbit polyclonal anti-Col1α1, α-SMA, JAK1, p-JAK1, STAT1, p-STAT1, STAT3, p-STAT3 and β-actin were diluted at 1:1000, rabbit polyclonal anti-LCK and SOCS1 (Abcam, USA) were diluted at 1:500, followed by incubation with secondary antibodies (1:10000, ZSGB-Bio, China) for 1 hour at room temperature. At last, the protein bands were detected by ECL-chemiluminescent kit (ECL-plus, Thermo Scientific).
Small RNA interference (siRNA) analysis
Small interfering RNA (siRNA) oligonucleotides against the LCK gene or scrambled sequences were designed and synthesized by Hanheng (Shanghai, China). The siRNA sequences were as follows: LCK-siRNA (sense, 5ʹ-GCAUCAAGUUGAA CGUCAATT-3ʹ and antisense, 5ʹ-UUGACGUUCAACUUGAUGCTT-3ʹ); Scrambled- siRNA (sense, 5ʹ-UUCUCC GAACGUGUCACGUTT-3ʹ and antisense, 5ʹ-ACGUGA CACGUUCGGAGAATT-3ʹ). HSC-T6 cells were transfected
with 1000 ng/ml LCK-siRNA or scrambled-siRNA and mixed with Lipo2000 transfection
reagent (Invitrogen, USA) according to the manufacturer's instructions. After 6 h, Opti-MEM was replaced by DMEM (10% FBS), and cells were activated by 10 ng/ml TGF-β1. The silencing efficiency was tested by RT-qPCR and Western blot.
Transfection with LCK plasmid
HSC-T6 cells were seeded in 6-well plates and cultured in DMEM (10% FBS). After adhering to the well, the medium was replaced with Opti-MEM (Gbico, USA) and cells were transfected with 1000ng/mL pEX-2-Control and pEX-2-LCK overexpression plasmid mixed with Lipo2000 transfection reagent (Invitrogen, USA) according to the manufacturer's instruction. After 6h, the Opti-MEM was changed to DMEM (10% FBS) contained 10ng/ml TGF-β1. After 24h, use MDI incubated for 48h.
Flow cytometry analysis
Cell cycle analysis kit (Beyotime) was used for cell cycle analysis. In simple terms, HSC-T6 cells were trypsinized, washed with cold PBS, and then, fixed in cold ethanol (75%, 3 mL) at 4°C overnight. Next, the cells were washed twice, treated with a 0.5 ml mixture (RNase and PI), and incubated for 30 min at 37°C in a dark place. A flow cytometer (Beckman, USA) was used to detect the cell cycle, and data were analyzed using FlowJo software (TreeStar, USA).
Co -Immunoprecipitation assay (Co -IP assay)
Co-IP has been successfully used to study the interaction of proteins. Co-IP contains of several steps, including preparation of protein extract, coupling a specific antibody to beads, purification of specific protein complexes. Purified protein compound can then be identified by western blot.
Data were represented as Mean ± SD. Statistical analyses were performed using a two-tailed unpaired t test or one-way ANOVA followed by the Newman–Keuls post hoc test (Prism 5 software, USA), p value < 0.05 was considered statistically significant.