Journal: Journal of Biological Chemistry

Article Title: Trypanosoma cruzi Induces the Reactive Oxygen Species-PARP-1-RelA Pathway for Up-regulation of Cytokine Expression in Cardiomyocytes

doi: 10.1074/jbc.m109.076984

Figure Lengend Snippet: FIGURE 3. ROS stimulates PARP-1 activation in cardiomyocytes infected by T. cruzi. A, PAR modification. AC16 cells were infected with T. cruzi for 0–48 h. Cell lysates (40 g of protein) were resolved by SDS-PAGE, and protein PAR modification was detected using anti-PAR antibody. B and C, enhanced PAR modification of cellular proteins is dependent upon PARP-1 and ROS activation. AC16 cells (wt and infected) were incubated with 10 M PJ34 (PARP-1 inhibitor), 50 M H2O2, or 0–500 M PBN for 12 h. Cell lysates (40 g of protein) were resolved by SDS-PAGE, and Western blotting was performed with anti-PAR antibody. D and E, PARP-1 is PAR- modified in a ROS-dependent manner. AC16 cells were infected with T. cruzi for 12 h (10 M PJ34 or 500 M PBN). Cell lysates were subjected to immunoprecipitation (IP) with anti-PARP antibody, and immunoblotting (IB) was performed with anti-PARP-1 and anti-PAR-1 antibodies. Densitometric analysis of PAR-modified PARP-1, normalized to -actin signal, is shown in E. Data (mean S.D.) are representative of three independent experiments (p 0.01; *, normal versus T. cruzi-infected; #, infected/PBN-treated versus infected/untreated). All membranes were re-probed with -actin antibody to validate equal loading of samples.

Article Snippet: Antibodies and Reagents—Monoclonal antibody against poly ADP-ribose (PAR) (ALX-804-220) was purchased from Alexis (SanDiego, CA).Monoclonal antibodies against PARP-1 (B-10, sc-74470) and NF- B p65 (F-6, sc-8008), and polyclonal antibodies against PARP-1 (H-300, sc-25780) and Lamin A/C (H-110, sc-20681), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Infection, Modification, SDS Page, Incubation, Western Blot, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Trypanosoma cruzi Induces the Reactive Oxygen Species-PARP-1-RelA Pathway for Up-regulation of Cytokine Expression in Cardiomyocytes

doi: 10.1074/jbc.m109.076984

Figure Lengend Snippet: FIGURE 7. PARP-1 activation contributes to a loss of mitochondrial mem- brane potential and enhanced ROS production in infected cardiomyo- cytes. AC16 cells (normal and infected) were incubated in presence or absence of 10 M PJ34 for 12 h. A, shown are the representative fluorescence micrographs of normal (panels a, d, and g), T. cruzi-infected (panels b, e, and h), and infected/PJ34-treated (panels c, f, and i) cardiomyocytes, stained with anti-PAR antibody (panels a–c, green) and MitoTracker Red (localizes to mito- chondria, panels d–f). Overlay images (panels g–i, yellow) demonstrate mito- chondrial localization of PAR in infected cardiomyocytes. B, shown are the representative fluorescence micrographs of JC-1-stained wt and infected car- diomyocytes (PJ34). Note the accumulation of green monomers in infected cardiomyocytes(panele)wasinhibitedbyPJ34(panelf).Overlayimages(pan- elsg–i)showthatamajorityofmitochondriafluorescedredinnormal(panelg) and PJ34-treated/infected cardiomyocytes (panel i), whereas mitochondria in infected cardiomyocytes fluoresced green (panels h). C, shown are fluorescent micrographs of wt and infected cardiomyocytes (PJ34) stained with MitoSOX Red (detects mitochondrial O2., panels a–c), and DHE (detects intra- cellular/cytosolic ROS, panels d–f, red) probes. Note that mitochondrial and cytosolic ROS were decreased by PJ34 treatment of infected cardiomyocytes.

Article Snippet: Antibodies and Reagents—Monoclonal antibody against poly ADP-ribose (PAR) (ALX-804-220) was purchased from Alexis (SanDiego, CA).Monoclonal antibodies against PARP-1 (B-10, sc-74470) and NF- B p65 (F-6, sc-8008), and polyclonal antibodies against PARP-1 (H-300, sc-25780) and Lamin A/C (H-110, sc-20681), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Infection, Incubation, Fluorescence, Staining