Small molecule inhibitors

Mithramycin (Tocris Bioscience) was dissolved as a 1 mg/mL stock solution in PBS, and frozen in aliquots. PHA-767491 and other small molecule inhibitors (Selleck Chemicals) were dissolved in DMSO as stock solutions. Small molecule stocks were stored at −80 °C for storage or −20 °C for experimental use, minimizing freeze-thaw cycles. For in vivo experiments, a 30 mg/mL stock solution of PHA-767491 (Adooq Bioscience) was made by dissolving the powder in a solution of 1 mg/10mL D-α-Tocopherol polyethylene 1000 succinate (MilliporeSigma) for oral gavage.

Small-molecule inhibitors were obtained from SelleckChem. siRNAs were obtained from GE Dharmacon. Cell lines were obtained from American Type Culture Collection (ATCC) and European Collection of Cell Cultures (ECACC) in 2010-2011 and maintained according to the supplier’s instructions as described in (19 (link),47 (link)). The MCF10A CDH1^-/- and MCF10A CDH1^+/+ isogenic cell lines, were obtained in 2014 from Sigma Aldrich, and maintained according to the supplier’s instructions. At sixth-monthly intervals and prior to storage, the identity of each cell line was confirmed by short tandem repeat (STR) profiling of 10 loci using the GenePrint 10 system (Promega). At monthly intervals, mycoplasma testing of cell cultures was carried out using MycoAlert Mycoplasma Detection Kit (Lonza).

Naive CD4⁺ T cells (CD62L⁺CD44⁻ or CD62L⁺CD44⁻FoxP3GFP⁻) were sorted from WT B6, Irf4^−/−, or Foxp3gfp reporter mice by a FACSAria flow cytometer. B6 APCs were prepared by depletion of T cells from B6 splenocytes with phycoerythrin–anti-CD3 (clone 2C11; BioLegend) and anti-phycoerythrin microbeads (Miltenyi Biotec, San Diego, CA), followed by brief treatment with 50 μg/ml mitomycin C (Fisher Scientific) before each experiment. For activation of T cells, naïve CD4⁺ T cells were added at 1×10⁵ cells/well in 96-well round bottom tissue-culture plates (Thermo Fisher Scientific), and stimulated with equal numbers of B6 APCs and 1 μg/ml soluble anti-CD3e mAb (clone 2C11; BioLegend). For some experiments cell cultures were supplemented with various cytokines (PeproTech, Rocky Hill, NJ) and small-molecule inhibitors (Selleck Chemicals). In some cases, naïve CD4⁺ T cells were labeled with CellTrace™ Violet reagent prior to stimulation. CD4⁺ T cells cultured for different days were collected and analyzed with flow cytometry, microarray analysis, Immunoblot, and quantitative real-time PCR, and ChIP.