pcna inh  (Tocris)

Journal: Frontiers in immunology

Article Title: ROS and DNA repair in spontaneous versus agonist-induced NETosis: Context matters.

doi: 10.3389/fimmu.2022.1033815

Figure Lengend Snippet: FIGURE 1 Oxidative DNA damage and repair drive NETosis: inhibition of early repair steps suppresses spontaneous NETosis while inhibition of late repair steps promotes it. (A) Immunoconfocal imaging confirms that neutrophils undergo low level spontaneous NETosis in media, while PMA, an oxidative burst inducer, promotes robust NETosis. Presence of DPI, an inhibitor of ROS generation, suppresses NETosis in both experimental conditions. DNA (DAPI, blue), MPO (pink). (B) NET DNA release from neutrophils incubated in media, or media with DPI or NAC indicates that ROS inhibitors suppress spontaneous NETosis (SYTOX; *, p<0.05 compared to control). (C) DNA release from neutrophils incubated in media or media with inhibitors (inh) of early stage oxidative DNA damage repair proteins APE (inh 1 or 2), PARP1 (inh 1 or 2) or LIG suggests that inhibition of these proteins suppresses spontaneous NETosis (SYTOX; *, p<0.05 compared to control). (D) DNA release from neutrophils incubated in media, or media with inhibitors of late stage repair proteins PCNA, Pol b or Pol d suggest that inhibition of these proteins promotes spontaneous NETosis (SYTOX; *, p<0.05 compared to control). (E) DNA release from neutrophils incubated with ROS inhibitors (DPI, NAC or FBS) shows that spontaneous NETosis is suppressed by ROS inhibitors regardless of the presence of inhibitors to PCNA, Pol b or Pol d in the media (SYTOX; *, p<0.05 compared to media treated control in each respective cluster). (F) Confocal imaging confirms that ROS inhibitor DPI suppresses spontaneous NETosis promoted by the inhibitors of PCNA, Pol b or Pol d. DNA (DAPI, blue). (G) ROS measurements by DHR123 assays show that neutrophils used in the experiments generate high levels of ROS when stimulated with PMA (+ve control) compared to non-activated neutrophils. Presence of inhibitors to PCNA, Pol b or Pol d does not alter the baseline ROS generation (Plate reader; *, p<0.05 compared to control). Serum was not added to the media unless otherwise stated. DNA repair inhibitors used were: APE inh 1 (CRT0044876), APE inh 2 (APE1 Inhibitor III), PARP1 inh 1 (BSI201), PARP inh 2 (PJ34) or LIG inh (L189), PCNA inh (T2AA), Pol b inh (AM-TS23) or Pol d inh (Aphidicolin). Fluorescence was recorded at 4-hour time points by SYTOX Green plate reader assays (n = 3 for all the experiments). The images are representative of 3 independent experiments, at 4-hour time points. Scale bar on images, 5 mm.

Article Snippet: ROS inhibitors (NOX inh Diphenyleneiodonium chloride, 1 mM DPI, Sigma), ROS scavengers (N-Acetyl-L-cysteine, 3 mMNAC, Sigma) and fetal bovine serum (1% (v/v) FBS, ThermoFisher Scientific) were added to the cells 1 hour before adding DNA repair inhibitors: Pol d inh (Aphidicolin, 50 mM, Sigma), Pol b inh (AM-TS23, 25 mM, Tocris), PCNA : Polymerase interaction inh or PCNA inh (T2AA, 25 mM, Tocris), APE inh 1 (CRT0044876, 125 mM, Sigma), APE inh 2 (APE1 Inhibitor III, 50 mM, EMD-Millipore), PARP1 inh 1 (BSI201, 100 mM, Sigma), PARP inh 2 (PJ34, 50 mM, EMD-Millipore), LIG inh (L189, 100 mM, Tocris).

Techniques: Inhibition, Imaging, Incubation, Control, Fluorescence

Journal: Frontiers in immunology

Article Title: ROS and DNA repair in spontaneous versus agonist-induced NETosis: Context matters.

doi: 10.3389/fimmu.2022.1033815

Figure Lengend Snippet: FIGURE 2 Confocal images showing that inhibiting the late stage oxidative DNA damage repair pathways induce spontaneous NETosis, and a diagram summarizing the regulation of spontaneous NETosis by various DNA repair proteins. (A, B) Neutrophils were treated with PCNA inhibitor (T2AA), immunostained and imaged by confocal microscopy. MPO (pink) colocalizing to DNA (DAPI blue) shows that PCNA inhibition promotes spontaneous NETosis (A). PCNA (red) is cytoplasmically distributed in the neutrophils but the inhibition of late stage DNA repair by inhibition of PCNA: polymerase binding leads to NETosis with PCNA present throughout the NET DNA (DAPI, blue) (B). Images for panels are representative of 3 independent experiments. Scale bar, 10 mm. (C) Diagram highlighting the proteins involved in the oxidative DNA damage repair pathways and their effects on spontaneous NETosis. Non-bulky adducts are repaired by BER. Baseline NETosis is suppressed by early DNA repair events that lead up to the chromatin decondensation/nick formation by APE1. Inhibition of late events of DNA repair promotes spontaneous NETosis. Proteins whose inhibition induces ( , blue arrow) or suppresses ( , red arrow) spontaneous NETosis are indicated. Overall, inhibition of early

Article Snippet: ROS inhibitors (NOX inh Diphenyleneiodonium chloride, 1 mM DPI, Sigma), ROS scavengers (N-Acetyl-L-cysteine, 3 mMNAC, Sigma) and fetal bovine serum (1% (v/v) FBS, ThermoFisher Scientific) were added to the cells 1 hour before adding DNA repair inhibitors: Pol d inh (Aphidicolin, 50 mM, Sigma), Pol b inh (AM-TS23, 25 mM, Tocris), PCNA : Polymerase interaction inh or PCNA inh (T2AA, 25 mM, Tocris), APE inh 1 (CRT0044876, 125 mM, Sigma), APE inh 2 (APE1 Inhibitor III, 50 mM, EMD-Millipore), PARP1 inh 1 (BSI201, 100 mM, Sigma), PARP inh 2 (PJ34, 50 mM, EMD-Millipore), LIG inh (L189, 100 mM, Tocris).

Techniques: Confocal Microscopy, Inhibition, Binding Assay