Anti α sma

ANE was purchased from Haw Yaun Vacuum Biochemical Technology Co., Ltd. (Taoyuan). Anti‐CTGF, anti‐α‐SMA, anti‐beta actin, and horseradish peroxidase‐conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). General chemicals used in this study were purchased from Sigma unless indicated otherwise.

For in vivo experiments, fresh frozen AAV transfected mouse liver tissues were sectioned to 5 μM and stained with DAPI (Beyotime) for 10 min. For in vitro experiments, primary HSCs were first fixed in 4% paraformaldehyde for 15 min and then permeabilized in 0.5% Triton X-100 for 15 min at room temperature. Cells were then blocked by 5% BSA and incubated with anti-desmin (5332, Cell Signaling Technology, 1:100 dilution) or anti-α-SMA (19245, Cell Signaling Technology, 1:200 dilution) antibodies at 4 °C overnight followed by appropriate secondary antibodies conjugated with Alexa Fluor 647. Finally, the cell nuclei were stained with DAPI. Images were visualized and captured by using a Leica SP8 confocal microscope.

Paraffin-embedded samples were cut into 4-μm sections and processed for IHC. Tissue sections prepared for antigen retrieval by microwave treatment in citrate buffer (pH 6.0) were incubated with anti-ELTD1 (Proteintech Group, Chicago, IL, USA), anti-αSMA (Cell Signaling Technology, Danvers, MA, USA), anti-vimentin (Cell Signaling Technology), and anti-CD34 (Santa Cruz, Dallas, Texas, USA) primary antibodies. Immunostaining was performed using the Envision System with diaminobenzidine (Dako Cytomation, Glostrup, Denmark). Images were viewed and assessed using a microscope (Eclipse 80i, Nikon, Tokyo, Japan). Assessments of the ELTD1 staining were scored by two experienced pathologists blinded to patient identity and clinical status. The staining analysis technique was performed as previously described 24 (link). In particular, the proportion score was assigned based on the proportion of positive cells and the intensity of staining. The proportion of positive cells was scored from 0% to 100%. The intensity of staining was scored from 0 to 3 (0, none; 1, weak; 2, intermediate; 3, strong). The final quantitation of each staining was obtained by multiplying the two scores. For patient survival analyses, the median was set as the cut-off to distinguish strong and weak staining of ELTD1.

Apatinib was contributed by HengRui Medicine Co., Ltd (Lianyungang, Jiangsu, China). The storage and usage of apatinib are implemented as previously reported.15 (link) (0.1% concentration of DMSO was used to dissolve apatinib at 100 mM for a stock solution and diluted into the work concentration with a culture medium.) Primary antibodies including anti-PARP, anti-Axin 2, anti-β-catenin, anti-cyclin D1, anti-vimentin, anti-α-SMA, anti-Survivin, anti-p-GSK-3β (ser9), anti-Ub, anti-p-VEGFR2, anti-VEGFR2, anti-p-Erk and anti-Lamin B1 were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-Actin, anti-Tubulin, anti-CD31 and anti-PCNA antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Alexa Fluor 488 goat anti-rabbit IgG was bought from Thermo Fisher Scientific (Waltham, MA, USA). Annexin V/PI (Propidium Iodide) staining kit was purchased from Beyotime Company (Nantong, Jiangsu, China). TUNEL assay kits were got from Vazyme Biotech Co., Ltd (Nanjing, Jiangsu, China). GTVisin^TM anti-mouse/anti-rabbit immunohistochemical analysis KIT was bought from Gene Company (Shanghai, China).