Collagen 1

Liver tissues from human liver cancer patients, Tg^TM4SF5 FVB/N mice, or C57BL/6 (WT or Tm4sf5^−/− KO) mice treated with or without DEN and in the absence or presence of TSAHC were processed for immunohistochemistry. Liver sections were fixed with 3.7% formaldehyde and embedded in paraffin. The fixed liver sections were deparaffinized and rehydrated. Antigen retrieval was performed with heat-induced epitope retrieval (HIER) using sodium citrate buffer (pH 6.0). Quenching and blocking were performed using 3% H₂O₂ in distilled water and 1% normal goat serum in phosphate-buffered saline (PBS). Antigens were stained using the avidin–biotin complex (ABC) method (VECTASTAIN Elite ABC HRP Kit, Vector) and were detected using 3,3′-diaminobenzidine (DAB) stain (Vector). Antibodies against TM4SF5 [17 (link)], collagen I (Acris Antibodies), pY⁷⁰⁵STAT3 (Cell Signaling Technology), normal rabbit or mouse IgG, α-SMA (Sigma-Aldrich), α-fetoprotein (AFP), CD34, Ki67, laminins (Abcam), α-l-fucosidase [FUCA (AFU)], and laminin γ2 (Santa Cruz Biotechnology) were used for immunostaining. Ten random images per slide were saved using a digital slide scanner (MoticEasyScan, Motic, British Columbia, Canada). The tissues were also processed with Masson’s trichrome as well as hematoxylin and eosin stains, as previously described [27 (link)].

Sub-confluent cells in normal culture media were transfected or infected with control or specific siRNA, shRNA vectors, or virus encoding the indicated molecules for 48 or 24 h, respectively, in the presence of vehicle or TSAHC [26 (link)] treatment. Animal liver tissues were harvested for whole-cell or tissue extraction using a modified RIPA buffer, as previously described [17 (link), 21 (link)]. The primary antibodies (generally at 1:1000 dilution) used included antibodies against laminins, pY³⁹⁷FAK, MICA/B (Abcam, Cambridge, UK), p-ERKs, ERKs (Cell Signaling Technology. Danvers, MA, USA), pY⁷⁰⁵STAT3 (Millipore, Billerica, MA, USA), β-actin, α-tubulin, SLAMF7 (CS1), MICA/B, STAT3, pY⁵⁷⁷FAK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FAK (BD Transduction Laboratories, Bedford, MA, USA), collagen I (Acris Antibodies, Herford, Germany), and TM4SF5 [17 (link)]. The TM4SF5_C-ter (epitope region of RKKQDTPH¹⁹⁷) antibody was custom designed (Pro-Sci, Poway, CA, USA).