Formalin

For colony suppression assays, cells were seeded at 7.5 × 10⁶ to 1 × 10⁶ cells the day before transfection. Cells were transfected with the indicated plasmid using Lipofectamine LTX and Plus Reagent (Invitrogen) for 24 hours. Media was replaced and cells were allowed to recover for an additional 24 hours, followed by plating 1.0 × 10⁵ to 2.5 × 10⁵ cells (depending on cell type) in triplicate in 6 well plates. Cells were selected in 800μg/mL G418 (Gibco) for pCMV-Neo-Bam plasmids, pcDNA3.1+ plasmids, and 100–400μg/mL hygromycin (Corning, 50 mg/mL) for pCMV empty vector and pCMV-human-PADI4 (Sino Biologicals) and cultured for 7–14 days depending on cell type. A non-transfected control was used for each cell type to observe selection. After observation of colonies, cells were fixed in 10% formalin (Epredia) for 10 minutes and stained with 0.5% crystal violet (Sigma) for 1 hour. Plates were imaged and single colonies were counted using ImageJ.

For clonogenic survival assays, cells were seeded at 1 × 10⁶ cells the day before treatment. Cells were treated with 100μM CB-839 for 24 hours. Media was replaced and cells were plated at a density of 1.0 × 10⁵ and 2.5 × 10⁵ cells in triplicate in 6 well plates. After observation of colonies, cells were fixed in 10% formalin (Epredia) for 10 minutes and stained with 0.5% crystal violet (Sigma) for 1 hour. Plates were imaged and single colonies were counted using ImageJ.