P4ha2

The total cell lysates were prepared in a high KCl lysis buffer with a complete protease inhibitor cocktail (Roche, Switzerland). The protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Equal amounts of protein samples were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were treated with 1% blocking solution in TBS for 1 h, and immunoblots were probed with the indicated primary antibodies at 4°C overnight. The primary antibodies used were P4HA2 (ab233197), Collagen I (ab34710), Collagen III (ab7778), and Collagen IV (ab6586) from Abcam (Cambridge, UK); Twist1 (25465-1-AP) from Proteintech Group (Rosemont, PA, USA); Snail (#3879), Slug (#9585), AKT (#4691), phosphor-AKT (#4060), PI3K(#4257), and phospho-PI3K(#4228) from Cell Signaling Technology (Danvers, MA, USA); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-47724) from Santa Cruz Biotechnology (Dallas, TX, USA). Then, the membranes were washed and incubated with HRP-labeled secondary antibodies (Molecular Probes, MA, USA). The fluorescence signals were detected using a BM Chemiluminescence Western Blotting kit (Roche, Basel, Switzerland). Densitometry quantification was calculated from triplicate assays and analyzed using ImageJ software.

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.