Penicillin streptomycin antibiotic mixture

Sf9 insect cells (ATCC # CRL-1711) were routinely propagated in TNM-FH medium (Gemini Bio-Products, West Sacramento, CA) supplemented with 0.1% (v/v) Pluronic 68 (Sigma, St. Louis, MO), 10% (v/v) fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA) and a Penicillin-Streptomycin antibiotic mixture (Life Technologies, Carlsbad, CA) at 27°C. Baculovirus amplification was performed in the presence of 3% (v/v) FBS. BTI-TN-5B1-4 (High Five—Vienna Institute of Biotechnology subclone) [17 (link)] cells were used for the expression of soluble influenza A hemagglutinin-based antigens and maintained at 27°C in HyClone SFX serum free media (Fisher Scientific, Hampton, NH) supplemented with Penicillin-Streptomycin antibiotic mixture.

Sf9 insect cells (ATCC # CRL-1711) were routinely propagated at 27 °C in TNM-FH medium (Gemini Bio-Products, West Sacramento, CA) supplemented with 0.1% (v/v) Pluronic 68 (Sigma, St. Louis, MO), 10% (v/v) foetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA) and Penicillin–Streptomycin antibiotic mixture (Life Technologies, Carlsbad, CA). For baculovirus amplification, the medium was switched to 3% (v/v) FBS. BTI-TN-5B1-4 (High Five – Vienna Institute of Biotechnology subclone) [17] (link) cells were used for expression of VLPs and maintained at 27 °C in custom modified serum-free IPL-41 medium (PAN-Biotech GmbH, Aidenbach, Germany) at 27 °C as described in [18] (link) supplemented with Penicillin–Streptomycin antibiotic mixture.

The root of Ilex asprella was obtained from Qingping herbal market and was identified by Guang-Xiong Zhou (Jinan University). Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin antibiotic mixture and fetal bovine serum (FBS) were purchased from Life Technologies (Grand Island, New York, NY, USA). Other chemical reagents were purchased from Sangon Biotech (Shanghai, China). Murine RAW 264.7 cells, purchased from the American Type Culture Collection, were cultured in DMEM with 10% FBS, and incubated in 5% CO₂ at 37 °C. The cells were seeded into 6-well plates in DMEM medium, supplemented with 10% FBS in 5% CO₂ at 37 °C. Tumor-associated macrophages (TAMs) from S180 tumor tissues (a syngeneic sarcoma) were obtained by using a published method [31 (link)]. TAMs were cultured under the same condition with the above macrophages.

Murine LLC cells, recently used in several high-profile preclinical studies31 (link)32 (link), were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. LLC cells were grown in RPMI 1640 (Sigma Chemicals Aldrich, Milan, Italy), supplemented with 10% fetal bovine serum and 1% penicillin streptomycin antibiotic mixture (Life Technologies, Inc.-Invitrogen, Grand Island, NY) in a humidified incubator (Heraeus, Hanau, Germany) at 37 °C with 5% CO₂ atmosphere. The LLC tumor model was generated by subcutaneous injection of 2 × 10⁶ cells into the right hind leg of C57BL/6 mice (Charles River Lab). Animals were housed in a limited-access animal facility. The animal room temperature and relative humidity were set at 22 ± 2 °C and 55 ± 10%, respectively. Artificial lighting provided a 24-h cycle of 12-h light/12-h darkness (7 a.m. to 7 p.m.).
All animal procedures were approved by the Shandong Cancer Hospital & Institute Ethical Committee Guide for the care and use of Laboratory Animals. The methods were carried out in accordance with the approved guidelines.

Apigenin was purchased from Enzo Life Science (Plymouth, PA, USA). α-Modified minimum essential medium (α-MEM), RPMI 1640 medium, and penicillin/streptomycin antibiotic mixture were obtained from Life Technologies, Inc. (Grand Island, NY, USA). Fetal bovine serum was obtained from Moregate BioTech (Bulimba, Australia). Recombinant murine M-CSF and recombinant human soluble RANKL (sRANKL) were from R&D Systems (Minneapolis, MN, USA) and Pepro Tech EC., Ltd. (London, UK), respectively.

Sodium phosphate (NaH₂PO4 and Na₂HPO4), ascorbic acid, alizarin red S, cetylpyridinium chloride, silver nitrate, ethylenediaminetetraacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, NP-40, and sodium deoxycholate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin-streptomycin antibiotic mixture and FBS were purchased from Life Technologies (Grand Island, NY, USA). DMEM and Hanks’ balanced salt solution (HBSS) were purchased from Welgene (Daegu, Korea). Phosphatase- and protease-inhibitor cocktails were purchased from Roche Diagnostics (Manheim, Germany). Antibodies specific for Bax, Bcl-2, and α-SMA were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Runx2 and Pit-1 were obtained from Proteintech Group (Chicago, IL, USA). Anti-β-actin and anti-SM22α were obtained from Sigma-Aldrich and Abcam (Cambridge, MA, USA), respectively. A polyclonal APE1/Ref-1 antibody generated by immunizing rabbits with recombinant human APE1/Ref-1 was prepared in our laboratory [26 (link)].

The human cancer cell line, MCF-7 cells (Invasive ductal carcinoma, Estrogen/Progesterone receptor positive cell line), MDA MB-231 (Adenocarcinoma, Estrogen/Progesterone/Her2+ receptor negative or triple cell line) and MCF 10A (non tumorous mammary cell line) were procured from National Centre for Cell Sciences (NCCS), Pune, India and American Tissue & Cell Culture (ATCC) were used in the present study. MCF-7 cells were cultured in DMEM-F12 (Life Technologies, USA) and MDA MB-231 in DMEM supplemented with 10% fetal bovine serum. MCF 10 A cells were cultured in DMEM/F12 media supplemented with supplemented with 5% horse serum (Life Technologies, USA) along with cholera toxin (100ng/ml), Epidermal growth factor (EGF 20ng/ml), hydrocortisone (500ng/ml), insulin (10μg/ml) (Sigma,USA) and 1% penicillin, streptomycin antibiotic mixture (Life Technologies, USA). Cells were incubated at 37°C and 5% CO₂ in specified media.