129S-PARP1tm1Zqw/J (PARP1-/-) mice were crossed with C57BL/6 mice to generate PARP1-/- mice on 129S/BL6 genetic background. PARP1-/- mice were bred with WT mice to generate PARP1+/- mice. All breeding pairs were purchased from Jackson Laboratory (Bar Harbor ME), and a standard PCR was performed to confirm the genotype of WT (PARP1+/+), PARP1+/- and PARP1-/- mice (S1A Fig).
T. cruzi (SylvioX10/4, ATCC 50823) was propagated by in vitro passage in C2C12 cells. The WT, PARP1+/- and PARP1-/- mice (all 129S/BL6 background, 6-weeks-old) were infected with T. cruzi (10,000 trypomastigotes/mouse, intraperitoneal). For some studies, C57BL/6 mice were infected as above, and then mice were given a treatment of 2-(dimethylamino)-N-(6-oxo-5,6-dihydrophenanthridin-2-yl)acetamide hydrochloride (PJ34, Sigma-Aldrich, St Louis MO). PJ34 is a cell-permeable, water-soluble, selective PARP1 inhibitor PJ34 (EC50 = 20 nM) and shown to be ~10,000 times more potent than the prototypical PARP inhibitor, 3-aminobenzamide [20,21]. PJ34 (12.5 mg/kg) was delivered intraperitoneally for three weeks (twice a week) beginning at 45 days’ post-infection (pi) when acute parasitemia was controlled. All mice were harvested at 150 days’ pi corresponding to chronic disease phase. Sera/plasma and tissue samples were stored at 4°C and -80°C, respectively.
Human cardiomyocyte cells (AC16, cat#SCC109, EMD Millipore, Burlington MA) were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium containing 12.5% fetal bovine serum (FBS). Human cervix epithelial cells (HeLa, ATCC, Manassas VA) were propagated in DMEM media supplemented with Earle's salts, 2 mM L-glutamine and 10% FBS. Cells were infected with T. cruzi (cell: parasite ratio: 1:5) for various studies.
“I have known one family so far where the husband died of appendiceal cancer at age 27 — a cancer so rare there are only six known cases of appendiceal cancer in the world right now,” she said. “Another family, he died at the age of 35. These cancers are taking them at 20, 30, 40 years old. We should be giving them the specialized care they need beforehand, not paying their widows after they’re dead.”