Powerplex 1.2 system

Genomic DNA was collected from LNCaP, C4-2 and JHU-LNCaP-SM using the DNA Easy Blood and Tissue kit (Qiagen, Valencia, CA) according to the manufacture's protocol. Genomic DNA was analyzed using the PowerPlex 1.2 system (Promega, Madison, WI) on an Applied Biosystem 3730XL genetic analyzer by the Fragment Analysis Facility at Johns Hopkins University. STR profiles were compared using the DSMZ database for known cell line reference profiles (http://www.dsmz.de/services/services-human-and-animal-cell-lines/online-str-analysis.html).

M14 (melanoma) was obtained from University of California, Los Angeles; Neuroblastoma IMR-32 (CCL-127) and breast carcinoma AU565 (CRL-2351) were obtained from American Type Culture Collection (ATCC). All the cell lines used were authenticated by short tandem repeat profiling with PowerPlex 1.2 System (Promega), and routinely tested for mycoplasma using MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza). The cells were cultured in RPMI1640 (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies) at 37°C in a 5% CO₂ humidified incubator. The cells were passaged three times after thaw.

PC3 (prostate), MCF-7 (breast), and A375 (melanoma) cancer cell lines were obtained from American Type Culture Collection via the UCSF Cell Culture Facility (San Francisco, CA, USA). All experiments were performed within 6–12 months of cell line authentication (using short tandem repeat (STR) analysis, PowerPlex 1.2 System, Promega) and mycoplasma testing (MycoAlert^™ mycoplasma detection kit, Lonza). PC3 and MCF-7 cells were cultured in low glucose DMEM, A375 cells in high glucose DMEM. In all cases, medium was supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units ml⁻¹ of penicillin and 100 μg ml⁻¹ of streptomycin and cells were cultured in a humidified chamber at 37 °C in 5% CO₂ in air.
Cells were incubated with U0126 (LC Laboratories) at doses of 50 μM for PC3, and 25 μM for MCF-7 and A375 cells. All treatments were performed over 48 hours with matching DMSO solvent controls (1:1000 in the culture medium) and U0126 was replenished every 24 hours. Treatment doses were determined as previously described (31 (link)) using the WST-1 cell proliferation assay (Roche) and doses were selected such that they induced a 50% drop in cell viability after 48 hours of treatment. In all cases, inhibition of the target at the selected drug dose was confirmed by probing for p-ERK levels using Western blotting (see below).

The cell lines LAN-1 and M14 were obtained from University of California, Los Angeles; NMB-7 from Dr. SK Liao of McMaster University, Hamilton, Ontario, Canada. SKNJB, SKNJC2, SKEAW were developed at MSK and all others in Table 1 were purchased from ATCC. They were authenticated by short tandem repeat profiling using PowerPlex 1.2 System (Promega), and periodically tested for mycoplasma using a commercial kit (Lonza). The luciferase-labeled tumor cell lines IMR-32-Luc and M14-Luc were generated by retroviral infection with a SFG-GFLuc vector.

Human cervical (HeLa, 2012), colorectal (HCT116, 2006 and 2016) and breast (MDA-MB-231, 2008) cancers cell lines were purchased from ATCC and grown in the recommended media. A HeLa cell line that stably expresses mitochondrially-targeted E. coli LigA (mitoLigA) (4 (link)) after transfection with the plasmid pCAG-mitoLigAYFP-Neo that encodes E. coli LigA fused at its N terminus to the LigIIIα mitochondrial targeting sequence and at its C terminus to EYFP. The telomerase-immortalized human fibroblast cell line HCA-Ltrt from Dr. Murnane (2010), was grown in DMEM/F12 medium with 10% FBS. Normal breast epithelium MCF10A cells from Dr. Rassool (2012) were grown using recommended medium and mixture of additives (Lonza/Clonetics Corporation) with 5% horse serum and 100 ng/ml cholera toxin. Cell lines lacking mitochondria DNA (Rho minus cells) were established as described (18 (link),19 (link)). The identity of commercially available cell lines was confirmed by STR profiling with the PowerPlex 1.2 System (Promega), most recently in 2016.