Brdu cell proliferation assay

Endometrioid endometrial cancer-1 cell lines were seeded at 2 × 10⁴ cells per well in 96-well plate and transfected with mutants or wild type of WHSC1 expression constructs before BrdU incorporation using BrdU Cell Proliferation Assay (Millipore, Temecula, CA, United States).

The bromodeoxyuridine (BrdU) cell proliferation assay (Millipore, Burlington, MA, USA) was performed to analyze the proliferation of M-1 and 2L3 cells. To compare the proliferation ability of M-1 and 2L3 cells, cells were cultured in 96-well plates (5 × 10³ cells/well) for 24 h. BrdU was added to the medium 2 h prior to the end of the incubation period. To explore the effect of mNGAL on 2L3 cell proliferation, 2L3 cells were first cultured in 96-well plates (2 × 10³ cells/well) for 16 h followed by mNGAL treatment for another 8, 24, and 48 h. BrdU was added to the medium 2 h prior to the end of the mNGAL incubation period. Cells were then fixed with fixing solution (200 μL/well) at 37 °C for 30 min, followed by incubation with BrdU detection antibody (100 μL/well) at 37 °C for 1 h and peroxidase conjugated goat anti-mouse antibody (100 μL/well) at 37 °C for 30 min. The cells were washed three times with Wash Buffer and blotted dry on paper towels between the above-described steps.
After incubation with TMB peroxidase substrate (100 μL/well) at 37 °C for 30 min in the dark, the reaction was stopped with stop solution (100 μL/well). Finally, the OD₄₅₀ of each sample was measured using an ELISA reader (Thermo Fisher Scientific).

Cells (1 × 10⁴ per well) were plated into 96-well plates and incubated at 37 °C overnight. The cells were treated with 10 μg/ml cetuximab and/or 0.5 μg/ml Myc-RUNX3 diluted in a cell culture medium. BrdU cell proliferation was measured in triplicate per plate according to the manufacturer’s instructions (BrdU Cell Proliferation assay, Cat # 2757, Millipore, Billerica, MA, USA). The absorbance at 450 nm was measured. All experiments were performed with triplicate samples.