Seaplaque agar

Proliferation assays were set up in a 96-well format with 100 cells plated per well. Proliferation was assessed every 24 hours over 6 days by the MTS assay, following manufactures instructions (Promega). Absorbance was read at 490 nm to determine proliferation and 650 nm to account for cellular debris on a BioTek Synergy Mx automated plate reader. Soft agar assays were performed as follows, 6-well plates were prepared with bottom agar composed of 3.2% SeaPlaque Agar (Lonza) in DMEM full media and allowed to solidify before 10,000 cells in top agar (0.8% SeaPlaque Agar in DMEM full media) were plated and allowed to solidify. DMEM full media with 2.5 µg/mL doxycycline was plated over the cells and cells were incubated under standard conditions (5% CO₂,37°C) for 2 weeks. Top media was removed and cells were fixed in 10% formalin (Fisher Scientific) containing 0.005% crystal violet (Sigma) for 1 hour at room temperature. Formalin was removed and colonies were imaged on a Leica S8 AP0 microscope. 12 images per cell line were taken and automated colony counts were done using ImageJ software. Results shown are a representative example of at least 3 independent experiments.

A layer of sea plaque agar (Lonza, Basel) at a concentration of 0.8% in culture media was placed into 6-well plates and allowed to solidify. A top layer was then placed into each well containing 10,000 cells in 0.48% agar in culture media and allowed to solidify. One mL of culture media was placed on top of the solidified agar with or without 2.5 μg doxycycline/mL of media. Plates were incubated for 2 weeks, with one mL of culture media added to every well after one week. After two weeks, the media was removed and cells were fixed in 10% buffered formalin (Thermo Fisher) containing 0.5% crystal violet for 2 hours. Each well was divided into four quadrants and photographed on a Leica S8 AP0 microscope. Colonies were quantified using ImageJ software and graphed using the Prism software package. Three wells were used for each condition, conducted in triplicate, giving 9 wells per condition and 36 quadrants for imaging. Two-tailed, unpaired t-tests with α = 0.05 were used to identify statistically significant differences in colony numbers (p < 0.05).