Etoposide

ProTAME was purchased from Boston Biochem (Cambridge, MA) as a 20 mM stock solution in DMSO, stored at −20°C and diluted in RPMI 1640 (Fisher Scientific, Loughborough, UK) immediately before use. Etoposide, doxorubicin and vincristine were purchased from Cayman Chemical (Cambridge, UK), reconstituted in DMSO and stored at −20°C.

Staurosporine was obtained from LC Laboratories (Woburn, MA), etoposide was from Cayman Chemical (Ann Arbor, MI), gemcitabine was from Toronto Research Chemicals (Toronto, ON, Canada), and nocodazole, puromycin and thymidine were from Sigma-Aldrich (St. Louis, MO). BrdU was obtained from BD Pharmingen (San Diego, CA). Polyclonal antibodies to PDZRN3 were generated and purified as described previously^{16 (link)}. Rabbit polyclonal antibodies to cyclin A2 (GTX103042) and to Mre11 (GTX118741) were obtained from GeneTex (Irvine, CA), those to cytochrome c (#21680) were from Signalway Antibody (College Park, MD), those to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 2275-PC) were from Trevigen (Gaithersburg, MD), and those to cleaved caspase-3 (#9661), to Ser¹⁵-phosphorylated p53 (#9284), to Ser⁴⁷³-phosphorylated Akt (#9271), and to Akt (#9272) were from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibodies to α-tubulin (T-9026), to p53 (#2524), to Ser¹³⁹-phosphorylated H2AX (GTX628789), and to BrdU (66241-1-Ig) were from Sigma-Aldrich, Cell Signaling Technology, GeneTex, and Proteintech group (Chicago, IL), respectively, and rabbit monoclonal antibodies to Ki-67 (RM-9106-R7) were from Thermo Fisher Scientific (Waltham, MA). Phosphatase and protease inhibitor cocktails were obtained from Roche (Branchburg, NJ).

MDA-MB-231 human breast cancer cells (American Type Culture Collection, Manassas, VA) were cultured in L-15 Leibovitz's Medium (Corning, Manassas, VA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA) and maintained at 37°C with atmospheric CO₂ according to the suppliers instructions. Purified recombinant cmvIL-10, hIL-10, IFNγ, IL-6 and anti-cmvIL-10, anti-hIL-10, anti-IL-10R, and anti-serpin E1/PAI antibodies were purchased from R&D Systems (Minneapolis, MN). Total Stat3, pStat3 (Y705), total Stat1, and pStat1 (Y701) antibodies were from Cell Signaling Technology (Danvers, MA). The Stat3 inhibitor was from Santa Cruz Biotechnology (Dallas, TX), the Jak1 and p38 MAPK inhibitors were from Calbiochem/EMD Millipore (Billerica, MA). Etoposide was from Cayman Chemicals (Ann Arbor, MI) and purified recombinant human EGF was from Peprotech (Rocky Hill, NJ). The HCMV strain AD169 (ATCC) was propagated in human foreskin fibroblasts (HFF, also from ATCC), maintained in Dulbecco’s modification of Eagle’s medium (Corning) containing 15% fetal bovine serum.

Benzo[a]pyrene (BaP; CAS no. 50-32-8; purity ≥96%), cisplatin (CAS no. 15663-27-1, crystalline) and ellipticine (CAS no. 519-23-3; purity ≥98%) were obtained from Sigma-Aldrich. Etoposide (CAS no. 33419-42-0; purity ≥98%) was obtained from Cayman Chemical. The BaP metabolite (±)-trans-7,8-dihydroxy-7,8-dihydro-BaP (BaP-7,8-dihydrodiol) that was used as a standard for HPLC was synthesised at the Biochemical Institute for Environmental Carcinogens using earlier published methods (Platt and Oesch, 1983 ; Yagi et al., 1977 (link)). Mass spectrometry data and high field ¹H NMR spectra (400 MHz) for BaP-7,8-dihydrodiol were in essential agreement to those published previously.

GC7 (Merck Millipore) was added for 24 hours to B cells (10 μM) on day 2 after B cell stimulation (unless otherwise indicated) or to Jurkat/NIH 3T3 cells (100 μM). 10 nM bafilomycin A1 (Cayman Chemical), 10 μg/ml cycloheximide (Sigma), or 100 nM Torin 1 (Cayman Chemical) were added to cells for 2 hours. 10 μM etoposide (Cayman Chemical) or 1 μM thapsigargin (Cayman Chemical) was added to cell culture for 6 hours, or 1 mM difluoromethylornithine (DFMO, Enzo Life Sciences) for 24 hours.