Camptothecin

500 cells were seeded in triplicate in 6-well plates. 24 hr after seeding, cells were treated with Etoposide (72 hr), Camptothecin (Santa Cruz; CAS 7689-03-4)(12 hr) and formaldehyde (Sigma; F8775) (12 hr) before washing out the drug and replacing cells with DMEM + FBS. Cells were allowed to grow for a total of 10 days before fixing with 4% paraformaldehyde and staining with cresyl violet. Colonies with more than 50 cells were scored. Surviving fractions were calculated and plotted. Data represents the mean of three replicate experiments. Survival fractions were calculated as [number of colonies formed after treatment / (number of cells seeded X PE)]. PE = plating efficiency and is calculated as (number of colonies formed/ number of cells seeded) X 100%. Camptothecin was dissolved in DMSO at 25 mM stock concentration.

Hydrogen peroxide (H₂O₂) was bought from MP Biomedicals, USA. Keratinocyte-SFM medium, RPMI 1640 medium, penicillin, streptomycin, fetal bovine serum and l-glutamine were purchased from GIBCO, Invitrogen, USA. Annexin V-Fluorescein isothiocyanate (FITC) Apoptosis Detection Kit I (BD Pharmingen™) and Flow Cytometry Mitochondrial Membrane Potential Detection Kit were bought from BD™ MitoScreen, Becton–Dickinson Biosciences, USA. Camptothecin (CPT) was purchased from Santa Cruz Biotechnology, CA, USA. Ammonium acetate was bought from Merck, Germany. Chloroform was bought from R&M Chemicals, UK. Phenol and Sodium dodecyl sulfate (SDS) were procured from Amresco, USA. Isoamyl alchohol was purchased from Fluka, Switzerland. Phusion High-Fidelity DNA Polymerase was procured from Finnzymes, Finland. PCR primers were from First Base Laboratories. QIAquick Gel Extraction Kit and QIAquick Nucleotide Removal Kit were bought from QIAGEN, Germany. DNA Polymerase I Large (Klenow) Fragment, restriction enzymes and T4 DNA Ligase were obtained from New England Biolabs (NEB), USA. dNTP mix was purchased from Promega, USA.

Mouse antibody against AIF and rabbit antibodies against p53, H2B, and Cyp40, respectively, were purchased from Santa Cruz Biotechnology; mouse antibody against CytC and rabbit antibodies against VDAC and CypA were from Abcam. Monoclonal mouse anti-E1 antibody was from Viral Antigens, and mouse anti-alpha tubulin was obtained from Sigma Aldrich (St. Louis, MO, USA). Secondary antibodies for immunofluorescence and Western blot analysis were from Dianova. The pan caspase inhibitor z-VAD-fmk was from PeptaNova (Sandhausen, Germany); PFTμ and α, staurosporine and camptothecin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). NIM811 was generously provided by Novartis (Basel, Switzerland). All other reagents were from Sigma Aldrich. Stock solutions were prepared in dimethyl sulfoxide (DMSO), stored at −20 °C and diluted in cell culture medium to their respective final concentrations directly before use. Final concentration of DMSO, which was employed as vehicle (solvent) control, never exceeded 0.1%.