Cytation 3 image reader

Cells were seeded into 12-well plates and treated for 72 h with the corresponding treatments (Table S1). For long-term treatments, cells were seeded into 6-well plates and split once per week at a ratio of 1:3. Relative cell numbers were assessed using crystal violet. Adherent cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde in PBS. After 20 min, cells were washed twice with PBS and then 0.1% crystal violet solution in distilled water was added and left on the cells for 30 min. Crystal violet was carefully discarded. Stained cells were washed with distilled water and left to air-dry overnight. Glacial acetic acid was added to dissolve the crystals and the absorbance was measured at 595 nm with a Cytation 3 Image Reader (Agilent). The values of vehicle-treated controls were set to 100%.

Cells were seeded at 60% confluency in 6-well plates and incubated in hormone-deprived medium for 72 h. Using the transfection reagent PEI MAX 40 K (Polysciences Inc. # 24765-100), cells were co-transfected with 50 ng of the Renilla luciferase transfection control plasmid pRL-CMV (Promega #E2261) and 1 μg of either the ERα reporter plasmid XETL (here referred to as ERE-Luc) [20 (link)] or the HIF1α reporter plasmid HRE-Luc [152 (link)] (a gift from Navdeep Chandel (Addgene #26731)). 24 h later, cells were harvested and seeded into 96-well plates. 24 h after seeding, cells were treated with the corresponding drugs for another 24 h (Table S1). For assays using HRE-Luc, cells were incubated in 1% O₂ using a hypoxia incubator during the drug treatments. For assays in HEK 293 T cells, 1 μg of the plasmid HEG0 [153 (link)], which expresses full-length ERα, was added to the PEI MAX transfection mixture mentioned above. In parallel, a group of cells were transfected with the plasmid pSG5 [154 (link)] as the empty vector control. Using the dual-luciferase detection kit (Promega #E1910), cells were lysed in the lysis buffer for 30 min, and luciferase activities were measured with Cytation 3 Image Reader (Agilent). Firefly luciferase activities were normalized to those of the Renilla luciferase.

In vitro cell transformation was evaluated by serial passage culture and soft agar assay. Juvenile chondrocytes were passaged up to 13 passages (57–67 days in total) with a seeding density of 10,000 cells/cm². Cells were observed every day and cell growth rate was calculated as doubling time. Cell transformation was assessed by detecting anchorage-free proliferation. Cultured cells isolated from P2 chondrocyte sheets of normal culture periods (2 weeks) and extended culture periods (3.5 weeks) were seeded in a soft agar gel with chondrocyte culture media with ascorbate at a density of 5,000 cells per well by using CytoSelect Cell Transformation Assay (CBA-140, Cell Biolabs, San Diego, USA). Fluorescent signal representing cell number was measured with a spectrofluorometer (Cytation 3 image reader, BioTek, Winooski, USA) on day 0 and day 8 according to manufacturer’s protocol. HepG2 cells (HB-8065, ATCC) in DMEM containing 10% FBS and 1% antibiotic-antimycotic were used as positive control for anchorage-free cell growth. Data are shown as averages of relative fluorescent units from duplicate or triplicate assays.