Fig 3. Expression of mtDNA replication machinery and POLG interaction with PARP1 in chagasic mice.
(A) Mice (WT, PARP1+/-, and PARP1-/-) were infected with Tc and sacrificed at 150 days’ pi. (A-F) Shown is RT-qPCR evaluation of myocardial levels of mRNAs for components of the mtDNA replication and transcription machinery in chronically infected and control mice. Data were normalized to GAPDH mRNA (n ? 5 mice/group, triplicate observations per mouse). (G-I) Heart homogenates were subjected to differential centrifugation to isolate mitochondria. Representative Western blot images for cardiac mitochondrial levels of POLG (G, n = 4 mice/group) and other proteins of mtDNA replication machinery (I) in WT and PARP1-/- mice are shown (loading control: COIV). Densitometry analysis was performed for Western blot gels representing n? 4 mice/group, and data were normalized to COIV levels (H). (J) Cardiac mitochondria from normal and chagasic WT mice (n = 5/group) were pooled and subjected to immunoprecipitation with anti-POLG antibody. Input (total), flow-through (unbinding), and POLG-binding fractions were used for Western blotting with antibodies against PARP1, Twinkle, and SSBP1. WB with anti-POLG antibody was performed to confirm high efficiency of POLG pull down by immunoprecipitation in all samples. The gel images are representative of three independent experiments. (K&L) PARP1-POLG binding and POLG activity in Tc-infected cells. HeLa cells were infected with T. cruzi (cell: parasite ratio, 1:5) for 24 h (controls: uninfected or treated with 50 ?M H2O2). Cells were used for cross-IP/WB with antibodies against POLG and PARP1 (K). HeLa cells (± T. cruzi) were treated with formaldehyde to cross-link proteins and DNA. After immunoprecipitation with PARP1 antibody, DNA was used for PCR amplification of mitochondrial 7SDNA and nuclear COIV DNA fragments. The GAPDH and 7S DNA in input samples were amplified to confirm equal loading of all samples (L). Data in K and L are representative of three biological replicates. Data in bar graphs are plotted as mean value ± SEM, and statistical significance are marked as *WT.Tc vs. WT, &PARP1-/-.Tc vs. PARP1-/-, and #WT.Tc vs. PARP1-/-.Tc (*,&,#p<0.05, **p<0.01).