Rhtgfbi

Whole cell lysates were prepared from cell lines with RIPA lysis buffer (100 mM Tris pH 7.5, 150 mM sodium chloride, 0.1% deoxycholate, 0.1% SDS, 50 mM NaF, Protease inhibitor cocktail (Roche), 2 mM PMSF, 2 mM sodium orthovanadate) combined with scraping and the lysates cleared by centrifugation. Standard Western blotting procedures were performed. The following primary antibodies were used for immunoblotting at a dilution of 1:1000: ZEB1 (3396, Cell Signaling Technology, Danvers, MA, USA), Full-length PARP (9532, Cell Signaling Technology), Hsp90 (sc-13119, Santa Cruz, Dallas, TX, USA) and β-actin (MABT825, MilliporeSigma). Treatments with rhTGFBI (R&D Systems, Minneapolis, MN, USA) or vehicle control (PBS) were performed at the specified doses for 24 h prior to harvesting lysates. All blots or gels derive from the same experiment and were processed in parallel. See Supplementary Information for unedited blots (Supplementary Fig. 8a–c).

Primary tumorsphere formation was assessed in cells grown under anchorage-independent conditions in methylcellulose. BT549 (15,000) or LM2-4 cells (10,000) were cultured in 0.9 mL of 1% methylcellulose/complete DMEM medium in ultra-low adhesion 24-well dishes (Corning, Corning, NY, USA) and cells cultured for 10−12 days. Primary tumorspheres were assessed by counting colonies consisting of at least 6 cells from 4 fields per well with a 10× objective. We measured self-renewal by collecting primary tumorspheres by dilution in at least 3 volumes of PBS, dissociating them with trypsin for approximately 10 min, and re-seeding in 1% methylcellulose before evaluating secondary colonies after 10 days. For treatment with rhTGFBI (R&D Systems), a single dose of 500 ng/mL was added only once, when initially embedding the cells, and compared against cells receiving the same volume of vehicle (PBS).