Hank s balanced salt solution hbss

Exenatide and GLP-1A-Cys were purchased from Anygen (Gwangju, Republic of Korea). Oligomeric deoxycholic acids (DOCAs) were synthesized from Mediplex (Seoul, Republic of Korea) with 4-methylmorpholine (4-MMP) (Sigma-Aldrich, St. Louis, MO, USA), dicyclohexylcarbodiimide (DCC) (Sigma-Aldrich), N-hydroxysuccinimide (NHS) (Sigma-Aldrich), lys(BOC)OMe and BOC-lysBOC-Osu, DOCA ethylenediamine [molecular weight (MW) of 434.7], bisDOCA ethylenediamine (MW of 937.4), and tetraDOCA ethylenediamine (MW of 1,942.9). For chemical conjugation of GLP-1A with oligomeric DOCAs, 6-maleimidohexanoic NHS, N,N-dimethylformamide (DMF), and triethylamine were purchased from Sigma-Aldrich. For in vitro experiments, Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), and Hank’s balanced salt solution (HBSS) were purchased from Corning Inc. (Corning, NY, USA). Krebs–Ringer bicarbonate buffer (KRBB) was purchased from Sigma-Aldrich. In addition, rabbit monoclonal anti-E cadherin (Abcam, Cambridge, UK), rabbit monoclonal anti-Rps20 (Abcam), anti-SLC10A2 rabbit polyclonal antibody (Bioss Antibodies, Woburn, MA, USA), and anti-rabbit immunoglobulin G–horseradish peroxidase secondary antibody (Sigma-Aldrich) were purchased for western blotting.

Extraction of stromal cells from VHL-HBs was adapted from Park et al. 2007. Briefly, sterile resected surgical specimens were harvested from VHL-HBs and were immediately submerged in warm media (1:1 Advanced MEM (Gibco, MA, USA): RPMI (Gibco. MA, USA) with 15% Horse Serum (Gibco USA), 5% Heat-inactivated Fetal Bovine Serum (Gibco USA), L-Glutamine (Gibco USA), Penicillin Streptomycin (Gibco USA). All methods henceforth performed in sterile conditions. Specimen washed 2x with Hanks Balanced Salt Solution (HBSS; Corning) and single-cell suspensions were established by mechanical disaggregation, manual trituration and enzymatic digestion utilizing Papain (Lyophyilzed, 15 U/mg activity, LS003119, Worthington Biochem, Lakewood, NJ) and DNase I (1 U/ul, D4263, Sigma USA) following manufacturer’s protocol. The cellular layer was isolated from contaminating red blood cells and cellular debris with lymphocyte separation medium (Corning) and density-gradient centrifugation. Cells were then washed 2x with HBSS, resuspended in media with the addition of 100 Units/ml of epoetin alpha (EPO) (2000 U/ml Amgen) and plated in tissue culture nonpyrogenic polystyrene treated plates (Corning).

Fexofenadine hydrochloride and emodin were purchased from Tokyo Chemical Industry (Tokyo, Japan). Dimethyl sulfoxide (DMSO), terfenadine, verapamil, Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, MEM non-essential amino acid solution (NEAA) and glutamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile and water were purchased from Fisher Scientific Korea (Seoul, Korea). emodin, emodin-8-O-β-d-glucoside, chrysophanol, chrysophanol-8-O-β-d-glucoside, physcion and physcion-8-O-β-d-glucoside isolated from R. acetosa were obtained from the pharmacognosy laboratory of the College of Pharmacy at Gyeongsang National University (Jinju, Korea) [27 (link)]. Fetal bovine serum (FBS), N-2-hydroxyethylpiperazine–N′-2-ethanesulfonic acid (HEPES) and Hanks’ balanced salt solution (HBSS) were purchased from Corning (Manassas, VA, USA). Penicillin–streptomycin, Opti-MEM and 0.25% (w/v) trypsin–EDTA were purchased from Gibco (Carlsbad, CA, USA). Phosphate buffered saline (PBS) was purchased from Welgene (Gyeongsan, Korea). An MDR assay kit (fluorometric) was purchased from Abcam (Cambridge, UK).