Rhtgf β1

The MC38 and CT26 cell lines were obtained from the Einstein Cytogenetics Facility cell line repository. The SKBR-3, MDA MB-468, OVCAR-3, OVCAR-4, and NCI HC322M cell lines were derived from the National Cancer Institute Developmental Therapeutics Program cell line repository. The Hepa1-6 cell line was obtained from the Marion Bessin Liver Center at the Albert Einstein College of Medicine. MC38-derived cell lines were cultured in RPMI (Corning) supplemented with 10% FBS. Hepa1-6 and CT26-derived cell lines were cultured in DMEM (Corning) supplemented with 10% FBS. Primary T cells were cultured in T cell medium: RPMI supplemented with 10% FBS, 100 U/mL penicillin-streptomycin, 10 mM HEPES, L-glutamine, 1 mM sodium pyruvate, MEM non-essential amino acids, and 55 µM β-mercaptoethanol. T cell medium was also supplemented with cytokines (Biolegend) as needed; 10 ng/mL rhIL-2, and variable concentration of rhTGFβ1 (Biolegend) as noted per experiment.

All cells were incubated at 37°C, 5% CO₂. Standard culture volumes for different plate formats were as follows: 10cm dish; 10 mL, 6-well; 2 mL, 12-well; 1 mL, 24 well; 0.5 mL, 96-well; 0.1 mL. iPSC were cultured in an in house alternative to commercial Essential 8 medium called OXE8 medium (35 ) on Geltrex™ (Gibco, #A1413201)-coated tissue culture dishes and passaged either as clumps using 0.5 mM EDTA in PBS, or using TrypLE™ Express (Gibco, #12604013). For 24 hours after TrypLE™ passaging, media was supplemented with 10 µM Y-27632 (Abcam, ab120129). Differentiation of iPSC into iPSC-Macs was carried out as previously described (35 , 36 (link)) culturing in OXM medium and with the final 7 day differentiation occurring with cells plated at stated densities. iPSC-derived microglia were cultured based on a previously publication (37 ) by differentiation of iPSC-Mac precursor cells (PreMacs) for 14 days on fibronectin (Sigma, #F4759-1MG) coated tissue culture plates in RPMI (ATCC formulation, ThermoFisher, #A10491-01) supplemented with 100 ng/ml rhIL34 (Peprotech, #200-34-100uG), 25 ng/ml rhM-CSF (Life technologies, #PHC9501), 50 ng/ml rhTGF-β1 (Biolegend, #781804) and 1% Pen/Strep, with feeding every 3 days.

Chondrocytes were stimulated with recombinant human (rh) TGF-β1 (BioLegend, San Diego, CA, USA), rhIL-1β (R&D Systems, Minneapolis, MN, USA), OA synovium-conditioned medium (OAS-cm), or a combination of these mediators, for time periods and dosages indicated in Figure legends. OAS-cm was obtained by culturing synovium from OA patients for 24 h, whereafter debris was removed by centrifugation at 300× g and medium was stored in aliquots at −20 °C until further use [48 (link)]. To inhibit ALK5 kinase activity, 5 µM SB-505124 (Sigma-Aldrich, Burlington, MA, USA) was used, dissolved in dimethyl sulfoxide (DMSO).