Becoming partly cloudy with isolated thunderstorms developing late. Low around 75F. Winds SSE at 10 to 15 mph. Chance of rain 30%..
Becoming partly cloudy with isolated thunderstorms developing late. Low around 75F. Winds SSE at 10 to 15 mph. Chance of rain 30%.
Updated: June 2, 2018 @ 3:58 pm
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.