Phendc3

HCT116 cells were seeded at 650 000 cells/well in six-well plates, 24 h prior to treatment. Along with 1 μl/well of lipofectamine 2000 (ThermoFisher), the ligands were then added to the media at final concentrations of 2 μM cPDS (carboxypyridostatin trifluoroacetate salt, Sigma-Aldrich, working solution 1 mM in water), 20 μM Phen-DC3 (Polysciences Inc., working solution 2 mM in DMSO) and 2 μM TmPyP4 (meso-5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine, Calbiochem, working solution 1 mM in water) and the cells incubated for 24 h at which point they were compared to vehicle-only treated cells. All treatments were performed in either triplicate (for cPDS) or duplicate (Phen-DC3 and TmPyP4), and were repeated on two different days (n = 2). Cells from each well were harvested in 1 ml of ice-cold PBS using a cell scraper. The cell volumes equivalent to 1/5 and 4/5 of a well of a six-well plate were kept for the total RNA and the total protein extractions, respectively. Centrifugation at 1000 RPM for 10 min was performed to isolate the cell pellets, which were then stored at –80°C until the lysis and the RNA and protein extractions were performed.

A549, HeLa, 293T and U2OS cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS), and 50 U ml⁻¹ penicillin and 50 ng ml⁻¹ streptomycin (Gibco). None of the cell lines used in this study are listed in the database of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee70 (link). The A549 cell line was a gift from the Bradner Lab at Dana-Farber Cancer Institute and was verified by short tandem repeat (STR) profiling. The HeLa and U2OS cells originated from the Jasin Lab at Memorial Sloan Kettering Cancer Center. The 293T cells were gifts from the Alt Lab at Boston Children’s Hospital. Cells were tested for mycoplasma contamination by the IDEXX Laboratories or using the Lonza Mycoplasma kit. Puromycin (Gibco) selection in A549 and 293T cells was at 1.0 μg ml⁻¹. Hygromycin B (Invitrogen) selection in A549 cells was at concentration (200 μg ml⁻¹). Cell synchronization of A549 cells was achieved by growing cells to confluence, reducing FBS to 0.1% for 72 h and releasing cells into DMEM with 20% FBS. A549 cells were treated at the indicated concentrations of pyridostatin (Sigma Aldrich), PhenDC3 (Polysciences), ME0328 (Selleck), KU0058948 (Axon MedChem) or Bleomycin (Santa Cruz Biotechnology) when indicated. All cells were cultured under normal oxygen conditions (21% O₂, 5% CO₂, 37 °C).

Glioblastoma cells (LN18, ATCC CRL-2610; U251-MG ECACC #;09063001 U87, SIGMA, #89081402-1VL) were grown in DMEM media (4.5 g/l glucose) supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were tested for mycoplasma contamination by PCR. Cells were incubated/exposed at 37 °C with: 20 µM PDS (Selleckchem S7444) or 20 µM cPDS (Sigma-Aldrich SML1176) or 10 µM PhenDC3 (Polysciences, #26000-1) for the indicated time, 100 µg/ml Puromycin (Sigma P8833) for 1 h, 500 µM or dose scale of TMZ for 24 h, 4 Gy or dose scale of γ-irradiation (Gammacell 40 Exactor).