Cultrex cushions

MDA-MB-231 cells (1000 cells per well) were cultured in 96-well plates in the presence of BV02 (1.0 μM; Sigma-Aldrich, Product # 01040), MPS1-IN-3 (0.5 μM; Sigma-Aldrich, Product # SML0898) or DPH (40 μM) and their 2D-proliferation was analyzed utilizing the phase-contrast analysis mode on an IncuCyte ZOOM (Essen Bio-Science; Ann Arbor, MI, USA). Briefly, phase-contrast images were taken at the start of treatment and at 60 h, at which their final phase-contrast readings were normalized to initial phase confluence and statistical comparisons between the indicated treatments were made.
The ‘on-top’ method of 3D-organotypic culturing was utilized as described.^{35 (link), 43 (link)} Briefly, MCF-7 and MDA-MB-231 cells (1000 cells per well) were cultured in 96-well plates on 50 μl Cultrex cushions (Trevigen, Gaithersburg, MD, USA) in growth media supplemented with 5% Cultrex. Where indicated, the cells were treated with MPS1-IN-3 (0.5 μM), DPH (20 μM), or Imatinib mesylate (10 μM; LC Laboratories, Woburn, MA, USA), The media/Cultrex solution was replaced on days in which bioluminescence was being quantified. Biolouminescence was measured longitudinally after the addition of d-luciferin (Gold Biotechnology, St. Louis, MO, USA) as described.^{35 (link), 40 (link), 43 (link)}

NMuMG or 4T1 derivatives (7,500 cells/well) were cultured onto Cultrex cushions (150 ml; Trevigen, Gaithersburg, MD) housed in 48-well plates that contained complete media supplemented with 5% Cultrex. Where indicated, the Cultrex cushions were rendered biomechanically rigid by the inclusion of type I collagen (3 mg/ml; BD Biosciences), and cells propagated under compliant or rigid 3D-culture conditions were incubated in the absence or presence of TGF-β1 (5 ng/ml) as indicated. Differences in organoid growth were monitored by bright-field microscopy and quantified using Image J64 (version 1.46), while variations in β-galactosidase expression were detected and visualized using the Beta-Galactosidase Staining Kit (Mirus Bio LLC, Madison, WI) according to the manufacturer's instructions. Additionally, 4T1 derivatives (400,000 cells/well) were also cultured for 60 hr on Cultrex cushions (400 μl) housed in 6-well plates in the absence or presence of the TβRI inhibitor, SB431542 (10 μM) as indicated. Afterward, the resulting organoids were using the Cultrex 3D-culture cell harvesting kit (3448-020-K; Trevigen) according to the manufacturer's recommendations, at which point total RNA was isolated and purified as described above.