Ly2109761

CD14⁺ cells were freshly isolated from healthy donor buffy PBMCs using CD14⁺ magnetic beads (Miltenyi Biotech, Catalog number 130-050-201) (Supplementary Fig. S1A). The purity of CD14 selection was greater than 95% (Supplementary Fig. S1B). The CD14^– fraction was viably frozen in 95% FBS and 5% DMSO using a temperature gradient and stored for 5 days at −80C. CD14⁺ monocytes were then cultured in 10% fetal bovine serum-RPMI (complete) media and the indicated cytokines at the following concentrations: GM-CSF (10 ng/mL; Milteny Biotech Cat # 130-095-372), IL6 (10 ng/mL; Miltenyi Biotech Cat # 130-093-929), TGFβ (500 pg/mL; R&D Systems Cat #240-B-002). In some experiments, the TGFβ type I receptor kinase inhibitor (LY2109761, 2 μM) obtained from Eli Lilly under a Material Transfer Agreement, or a pan TGFβ-neutralizing antibody 1D11 (1 μg/mL; R&D Systems Cat #MAB-1835) were added to complete media.

All cell lines were obtained from American Type Culture Collection (Manassas, VA) and cultured in the recommended media in a humidified 5% CO₂ incubator at 37°C. To generate MDA231-Alk5^TD, -Alk5^KR, and -vec, retroviruses encoding TβRI(Alk5)^T204D, Alk5^K232R (3 (link)), or the empty pBMN-I-GFP vector were produced by transfecting Ampho-Phoenix cells and then utilized for transduction, followed by green fluorescent protein (GFP) selection. The miR-181a/b and MSH2 expression plasmids were constructed and described elsewhere (18 (link), 19 (link)). The BRCA1 expression construct was kindly provided by Dr. Jeffrey D. Parvin (Ohio State University). The ATM expression construct (23 (link)) was obtained from Addgene (Cambridge, MA). Plasmid constructions and additional reagents are described in Supplementary Material. Cell transfection, reporter assays, production of viruses, as well as infection and selection of transduced cells were carried out as previously described (19 (link)). Recombinant human TGFβ1 was purchased from R&D Systems (Minneapolis, MN). The type I/II TGFβ receptor inhibitor LY2109761 was provided by Eli Lilly and Company (Indianapolis, IN). ABT-888 was purchased from ChemieTek (Indianapolis, IN). 4-Amino-1,8-naphthalimide (ANI), doxorubicin, methyl methanesulfonate (MMS), and 6-thioguanine (6-TG) were purchased from Sigma (St. Louis, MO).