Collagen gel

hTERT-HM^Tet-3G cells (2.5 × 10⁵) cultured overnight in phenol red free DMEM/F12 complete growth medium were infected with ERΔ7 lentiviral particles in the presence or absence of Dox (500 ng/ml). After 48 h, cells were trypsinized and seeded into collagen gel and contraction assay was carried out following the manufacturer's protocol (Cell Biolabs Inc. San Diego, CA, USA). In brief, collagen lattice was prepared by mixing 2 parts of cell suspension and 8 parts of cold collagen gel solution to achieve 1.5 × 10⁵ cells/well. 0.5 mL of the cell-collagen mixture was added per well in a 24-well plate and incubated for 1 h at 37 °C to allow gelling. 1.0 mL of phenol-red free DMEM/F12 containing 2% charcoal-stripped FBS, 1% antibiotic-antimycotic, supplemented with or without Dox (500 ng/mL) and E2 (100 nM) was added over the cell-collagen matrix. The gels were gently released after 20 h and the area of the lattices were measured periodically from 4 to 36 h. Images of the floating gels were captured and digitized using a flatbed scanner (Hewlett Packard) and the mean gel area (cm²) was measured using IMAGEJ software. No Dox treatment served as control.