Crl 1651

Polyacrylamide (PA) gels of varying elastic moduli (E = 10 and 40 kPa) were fabricated as previously reported⁴⁵ . Gel stiffness was modulated by mixing acrylamide and bisacrylamide according to specifications reported in a well-established protocol⁴⁶ . Monkey kidney fibroblast cells (CRL-1651, ATCC, Manassas, VA) were plated sparsely on PA gels (2500 cells/cm²) and immersed in media comprised of Dulbecco’s Modified Eagle’s Medium (Corning, Corning, NY) (4.5 g/L glucose, 4 mM L-glutamine) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and 1% Penicillin Streptomycin (Corning, Corning, NY).

COS-7 (ATCC^® CRL-1651™) cells were grown in DMEM-high glucose supplemented with 10% heat inactivated fetal bovine serum, 1 mM sodium pyruvate, 2 mM l-glutamine and 1% penicillin–streptomycin at 37 °C in a 5% CO₂ incubator. All reagents used were from Gibco™ (Invitrogen, Carlsbad, CA). For transfection, COS-7 cells were plated into six-well culture plates and then transfected with the pDisplayAcGFP-PkDBPαII plasmid DNA (1 µg per well) using 10 µl Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA) in serum-free incomplete DMEM and grown at 37 °C in 5% CO₂. After 24 h, the transfection medium was replaced with complete DMEM-high glucose, and the cells were incubated for another 24 h. The transfected COS-7 cells were used in the erythrocyte-binding assay.

COS-7 cells were obtained from the ATTC (ATCC® CRL-1651™). COS-7 is a derivative of CV-1(ATCC^® CCL-70™), a cell line established from the kidney of an African green monkey that was transformed with an origin-defective mutant of SV40 [25 (link)]. Dulbecco’s modified Eagle medium (DMEM) supplemented with 100 U of penicillin, 100 μl of streptomycin per ml, (Sigma-Aldrich S.r.l., Milano, Italia) and fetal bovine serum (FBS) (10%) was used as maintenance medium for the cell line. The cells were incubated at 37 °C in the presence of 5% CO₂ and propagated at a ratio of 1:4 or 1:8.

Cell viability assays were performed on COS-7 cells (ATCC^® CRL-1651^™), a fibroblast-like cell line derived from green African monkey kidney, and on human THP-1-derived macrophages (ATCC^® TIB-202^™) harvested in 96 well plates at a cell density of 10,000 cells/well. Macrophages were obtained by differentiation of THP-1 cells using 50 nM phorbol-12-myristate-13-acetate (PMA, Sigma Aldrich Chimie, Saint Quentin Fallavier, France) for 72 h. Cells were incubated with feddeiketone B (1), 2,3-Dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone (2), syringylglycerol (3), or DANA (Sigma) for 2 or 3 h at a concentration range from 0.1 to 1 µM. Medium was removed and cells were incubated in obscurity for 4 h at 37 °C with a 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (MTT, Sigma, 5 mg/mL) diluted 1:6 in PBS. After incubation, medium was removed, wells washed with PBS, and 100 µL Dimethyl Sulfoxide (DMSO) (Sigma Aldrich, Darmstadt, Germany) were added to each well to solubilize formazan crystals. After 5 min agitation at room temperature, cell viability was assessed at 570 nm with Infinite F200 Pro (TECAN) hardware using the Magellan software.

sCT (H-2260, Bachem), hCT (H-2250, Bachem), ¹²⁵I-(Tyr22)-sCT (NEX423, Perkin Elmer) and ¹²⁵I-(Tyr12)-hCT (NEX422, Perkin Elmer). U2OS CALCR and COS-7 1375 cell lines: U2OS CALCR cells (PathHunter U2OS CALCR β-arrestin cell line, DiscoverX: 93-0566C3) cultured in MEM Eagle 1× (30-2003, ATCC) supplemented with 10% Fetal Bovine Serum (FBS) (F2442, Sigma-Aldrich), 1% Penicillin-streptomycin (P/S) (10376-016, Invitrogen), 300 μg/mL Hygromycin B (10687-010, Invitrogen) and 800 μg/mL Geneticin (10131-019, Invitrogen).
Cos-7 CT_(a)R cell line: parental Cos-7 cell line (CRL-1651, ATCC) with a stable transfection of a pEF-IRESpuro6 expression vector [21] (link) containing a cDNA insert for a cMyc-hCT_(a)(Leu variant) receptor construct (The stable transfected Cos-7 cell line was provided by Monash Institute of Pharmaceutical Sciences, DBB number: Cos-7 1375.10). The Cos-7 CT_(a)R cells were cultured in DMEM (21063-029, Invitrogen) supplemented with 5% FBS, 1% P/S and 10 μg/mL Puromycin (P8833, Sigma-Aldrich). All live cells experiments were incubated at 37°C in a humidified incubator with atmospheric air supplemented with 5% CO₂ unless otherwise specified.