Itga5

The cellular expression of the six candidate proteins were verified experimentally via Western Blot analysis. Total cell lysates were obtained using RIPA buffer (Thermo Fisher Scientific, United States). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, United States). The membranes were blocked in PBS, 10% (w/v) skim milk for 1 h in phosphate buffer saline-Tween 20 (PBS-T), and incubated for 3 h at RT in 5% milk in PBS-T with primary antibodies: CDH2, EGFR, ITGA3, ITGA5, ITGB1, and CALR (Abcam, Cambridge, United Kingdom). Then, after washing, the PVDF membrane was incubated with secondary antibody (The Jackson Laboratory, United States) for 40 min at RT. Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific, United States) was used for visualization.

Fibroblasts/10 mg tissue samples were lysed and western blot was preformed as previously described [17 (link)]. For integrin A5 expression levels, proteins were extracted directly from the tissue sample (10 mg, following biopsy) and from the cell line culture flasks during regular cell passages (between passages 3–8, at the normal proliferating phase).
The following rabbit/mouse anti-human antibodies were used: proliferating cell nuclear antigen (PCNA) (Sc-7907), pIκBα (Santa Cruz biotechnology, USA, Sc-8404), caspase-3 (#9665), cleaved caspase-3 (#9664), Phospho - signal transducer and activator of transcription 3 (pSTAT3 Tyr-705, #9145), pSTAT3-ser727 (#9134), STAT3 (Cell Signaling Technologies, USA, #9139), GAPDH (ab9484), ITGA5 (Abcam, USA, EPR7854) and alpha-Tubulin (Sigma, USA, T5168). Bound antibodies were visualized using Goat peroxidase-conjugated secondary antibodies (Millipore, USA, anti-Mouse IgG #AP308P and anti-Rabbit IgG #AP307P) followed by enhanced chemiluminescence detection (Millipore, USA). Optical densities were visualized and measured as arbitrary units by LAS3000 Imager (Fugifilm, USA). Results were normalized to Tubulin and GAPDH using Multi-gauge V3.0 program (Fugifilm, USA).

HCC samples and the corresponding adjacent nontumour liver tissues were collected from adult patients with HCC who underwent curative resection at the Tongji Hospital of Tongji Medical College (Wuhan, China). A preoperative clinical diagnosis of HCC was based on the diagnostic criteria of the American Association for the Study of Liver Diseases and haematoxylin & eosin staining of the samples was also performed. All of the samples were selected with distinctive pathologic diagnosis and none of the patients received any preoperative chemotherapy or radiotherapy. These tissues were stained for E‐cadherin (Cell Signaling Technology, 3195), MMP10 (Abclonal, A3033), P4HA2 (Abcam, ab233197), ITGA5 (Abcam, ab150361), MMP9 (Cell Signaling Technology, 13667), MT1X (Proteintech, 17172‐1‐AP) and SPP1 (Abclonal, A1499) expression. Furthermore, negative control for IHC staining was shown in Figure S8. IHC staining was performed using the Dako Envision Plus System (Dako) according to the manufacturer's instructions. The IHC staining intensity was scored as 0 (negative); 1 (weak); 2 (medium); 3 (strong). The percentage of positive cells was scored from 0 to 4 (0%, 1%‐25%, 26%‐50%, 51%‐75%, 76%‐100%). Overall score ranging from 0 to 12 was calculated by multiplying the above two scores, resulting in a negative (0‐3) staining or a positive (4‐12) staining for each example.