3 brpa

3-BrPA, verapamil (VRP), paclitaxel, MTT and rhodamine 123(Rh123) were purchased from Sigma-Aldrich (Deisenhofen, Germany). Doxorubicin (Dox) and epirubicin (EPI) were purchased from Zhejiang HISUN Pharmaceuticals Co (Zhejiang, China). Daunorubicin was supplied by National Institute for the Food and Drug Control (Beijing, China). Mouse anti-ABCB-1/P-gp was obtained from Santa Cruz (CA, USA). Other antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor 488 goat anti-mouse IgG (H+L) was purchased from Life Technologies (Gaithersburg, MD, USA).

The cell lines used in this research were TE-1, TE-11, ECA-109, KYSE150 and HEEC, which were purchased from Shanghai biology institute (Shanghai, China). 10% fetal bovine serum (GIBCO, USA) was added to culture media that containing 2 mM l-glutamine and 1% penicillin/streptomycin (Solarbio, China). Cells were grown in DMEM Medium (Trueline, USA) and maintained in a 5% CO₂, at 37 °C incubator. This study was in agreement with the Declaration of Helsinki. The AKT inhibitor LY294002 (25 μmol/L, S1105, Selleck, USA) and glycolytic inhibitor 3-BrPA (20 μmol/L, 1113-59-3, Sigma, USA) were dissolved in DMSO (D2650, Sigma, USA) and used to culture cells.

SF cultures were established by explant growth of small arthroscopic biopsy fragments of synovial tissues obtained from the knee of patients without previous joint disease at elective arthroscopy for minor traumatic lesions, or patients with RA or osteoarthritis (OA) at the time of prosthetic replacement surgery. Human adult pulmonary fibroblasts (HPF) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). Normal skin dermal fibroblasts (DF) were established by explant growth of skin biopsies obtained from healthy individuals during minor skin surgery. All fibroblast cultures were maintained in 10% fetal calf serum (FCS)-DMEM medium (Lonza, Verviers, Belgium). Patients signed a written informed consent, and the study was approved by the Ethics Committee of Hospital 12 de Octubre, Madrid, Spain. All methods involving humans were performed in accordance with the relevant guidelines and regulations.
Where indicated, cells were treated for 8 h with inhibitors 40 mM 2-DG, 25 μM 3BrPa, or 6 μM OL (Sigma-Aldrich, Madrid, Spain).

We have previously generated a human papillomavirus (HPV) E7-expressing tumorigenic cell line, TC-1 [17 (link)], and a firefly luciferase-expressing TC-1 cell line, TC-1-luc [2 (link)] (GenBank Accession LC456627.1). The H-2Db-restricted HPV16 E7aa49–57 peptide, RAHYNIVTF, was synthesized by Macromolecular Resources (Denver, CO) at a purity of ≥80%. PE-conjugated anti-mouse CD8a (clone 53.6.7), FITC-conjugated rat anti-mouse IFN-γ (clone XMG1.2) antibodies were purchased from BD Pharmingen (San Diego, CA). PE-conjugated, HPV16 E749–57 peptide loaded H-2Db tetramer was provided by NIAID tetramer core facility (Atlanta, GA). 3-BrPA, Benzo(a)pyrene (B(a)P, 99% pure), and tricaprylin were purchased from Sigma-Aldrich (St. Louis, MO).