Claudin 7

Paraffin-embedded tissue blocks were cut into 4-µm sections. Immunohistochemical analysis was performed using a Ventana XT automated stainer (Ventana, Tucson, AZ, USA), with antibodies against β-catenin (1:200, BD biosciences), E-cadherin (1:1600, Abcam), and Claudin-7 (1:200, Sigma). The percentage of nuclear β-catenin staining was evaluated and categorized as negative, weak, or high, based on percentage of tumor cells with nuclear expression (0%, 1–29%, or ≥30%, respectively). The H-score method, which assigns an immunohistochemistry (IHC) H-score to each case on a continuous scale of 0–300 based on percentage of cells at different staining intensities, was used for the interpretation of Claudin-7 and E-cadherin results. Membranous staining was scored as follows: 0 for “no staining,” 1+ for “weak staining visible at high magnification,” 2+ for “intermediate staining,” and 3+ for “strong staining”. The percentage of cells at different staining intensities was determined by visual assessment, with scores calculated using the following formula: 1(% of 1+ cells) + 2(% of 2+ cells) + 3(% of 3+ cells)^{15 (link)}. Samples were then classified as having low (H-score ≤ 200) or high (H-score > 200) protein expression.

Oridonin (No: 1126YA13, purity more than 98%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., in China. A 100 g/L stock solution of Oridonin was diluted in 100% dimethyl sulfoxide (DMSO) and stored at 4 °C in darkness. A serial dilution was made in 100% DMSO that was 1000 times more concentrated to allow for a 1:1000 dilution with EM to create a serial dilution with a final DMSO concentration of 0.1%. HepG2-Luciferase cells were purchased from Shanghai Biomodel Organism Science & Technology Development Co., Ltd. Cell Tracker CM-Dil (No: 1,583,101) was purchased from Shanghai Qianchen Biological Technology Co., Ltd. RPMI1640 culture solution (No: 11,875,093) was purchased from Invitrogen. Annexin V-FITC (No: 556,547) and Matrigel (No: 354,234) were purchased from BD Pharmingen. Calcein-AM (No: 56,496), MTT (No: M5655), penicillin, streptomycin, DMSO, and tricaine methanesulfonate (MS-222) were obtained from Sigma. Bevacizumab (Avastin®) and Trizol (No: 11,667,165,001) were purchased from Roche. Anti-CD31, claudin-1 (No: 81,796), claudin-4 (No: 37,643), and claudin-7 (No: 17,670) were purchased from Sigma. Recombinant RNase (No: 2313A), Reverse TranScriptase M-MLV (No: 2641A), and SYBR Premix Ex TaqTM (No: RR420) were purchased from Takara. The other chemicals used in this study were of analytical grade.