Fig. 2.

Minocycline inhibits of PARP-1 activity in neurons. (A) Photomicrographs show immunostaining for poly(ADP-ribose) (PAR, Left), and nuclei identified in the same fields with propidium iodide (Right). Neurons were fixed for staining 15–30 min after a 10-min incubation with 75 μM MNNG. PAR formation was suppressed by the PARP inhibitors DPQ (25 μM) and PJ34 (100 nM), and by minocycline (Mc, 100 nM). (Scale bar, 20 μm.) Representative of three independent studies. (B) Western blot of PAR formation in neurons harvested 15 min after a 10-min incubation with 75 μM MNNG alone or in combination with the designated concentrations of DPQ or minocycline. Control wells received medium exchanges only. PAR is seen as a discrete, major band at 116 kDa, corresponding to PAR formation on PARP-1 itself, and a smear of other bands resulting from PAR formation on other proteins. β-actin bands indicate relative protein loading in each lane. (C) Quantification of PAR Western blots. Data are means ± SEM of three independent experiments, each performed in triplicate. ∗∗, P < 0.01 compared to MNNG alone. (D) NAD⁺ content in neurons harvested after 30-min incubation with 75 μM MNNG. Data are means ± SEM of three independent experiments, each performed in triplicate. ∗∗, P < 0.01 compared to MNNG alone. (E) DNA damage in neurons visualized with the PANT method, conditions as in A. The increase in PANT-labeled neurons induced by MNNG was not blocked by DPQ or Mc.