Claudin 2

Total protein was extracted from rat ileums, and the concentration of total protein was determined by BCA Protein Assay kit (TransGen, Beijing, China). Western blotting was performed with the following primary antibodies: ACSL4 (Abcam), GPX4 (Abcam), FTH1 (Abcam), ZO-1 (Abcam), occludin (Zymed Laboratories), claudin-1 (Santa Cruz Biotechnology), and claudin-2 (Zymed Laboratories). β-Actin (Sigma) was used as loading control. For protein quantification, the density of Western blot bands was measured by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).

Representative paraffin blocks - defined as those with the largest amount of viable and anaplastic tumor - for each tumor were selected. Immunohistochemistry was performed on the tumor tissues and the neonatal ependyma and choroid plexus. The immunohistochemical reactions were performed on 3 μm sections obtained from the FFPE blocks. After the deparaffinization steps, the slides were treated in a microwave oven in Target Retrieval Solution (S1699 from DAKO, Carpenteria, CA, USA) for 30 min for heat-induced epitope retrieval. The immunohistochemical reactions were performed in an automated Ventana ES Immunostainer System (Ventana Medical Systems Inc., Tucson, AZ, USA) with the solutions and steps according to the manufacturer. The following antibodies and dilutions were used: claudin-1 (1:80, Zymed, #18–7362), claudin-2 (1:20, Zymed, #18–7363), claudin-5 (1:120, Zymed, #18–7364), claudin-7 (1:100, Zymed, #34–9100), E-cadherin (1:500, Dako #M3612), N-cadherin (1:300, Abcam #12,221), occludin (1:250, Invitrogen, Carlsbad, CA, USA ) and vimentin (1:300, Dako #M0725). The slides were counterstained with Mayer’s hematoxylin (Zymed, South San Fransisco, CA, USA). Positive controls and negative control tissues (with the omission of the primary antibodies) were included in every run.

IL-6 was procured from Sigma. Antibodies to occludin, ZO-1, claudin-2, MLC-2, and MLCK were purchased from Zymed Laboratories; the other antibodies were purchased from cell signaling. Alexa 594 secondary antibodies and DAPI (4#,6#-diamidino-2-phenylindole) were purchased from Molecular Probes. SYBR-Green PCR Master Mix is ABI (catalog number 43049155; Applied Biosystems). The standard of liquiritin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Nodakenin, quercitrin, arctiin, matairesinol, and icariin were purchased from Chemfaces (Hubei, China). Arctigenin was purchased from MUST (Chengdu Must Bio-Technology Co., Ltd., Chengdu, China). Angoroside C, neolicuroside, (8S, 8′′R)-8-(4-hydroxy-3-methoxybenzyl)-8′′-(3′,4′-dimethoxybenzyl)-γ-butyrolactone 4-O-(β-D-glycopyranoside), and emodin 8-O-β-D-glucopyranoside 8 were obtained from Chungnam National University (Daejeon, Korea). The purity of the standards was all above 95%. HPLC grade acetonitrile was purchased from J. T. Baker Inc. (Philipsburg, NJ, USA) and acetic acid purchased from JUNSEI (Junsie Chemical Co., Ltd., Tokyo, Japan). Ultrapure water was prepared by Puris-Evo-UP Water System with Evo-UP Dio VFT and Evo-ROP Dico20 (Mirae ST Co., Ltd., Anyang, Gyeonggi-do, Korea). Ultrapure water (UW) was prepared with a resistivity of 18.2 MΩ cm⁻¹ (Puris, Esse-UP Water System, Mirae ST Co., Anyang, Korea).