Journal: The Journal of Neuroscience

Article Title: Interaction of nNOS with PSD-95 Negatively Controls Regenerative Repair after Stroke

doi: 10.1523/JNEUROSCI.1305-14.2014

Figure Lengend Snippet: HDAC2 in NSCs is a mediator for the role of neuronal nNOS–PSD-95 association in regulating the fate of NSCs. A, B, Immunoblots showing HDAC2 levels (n = 4; A) and bar graph showing the percentage of newborn neurons (n = 3; B) in the NSCs infected by LV-HDAC2-shRNA, LV-control-shRNA, AD-HDAC2-Flag, or AD-Flag. C, Bar graph showing the percentage of newborn neurons in the DG of rats infected by AD-HDAC2-Flag or AD-Flag (n = 9). These rats were subjected to MCAO at 24 h after virus infection and were killed for BrdU/DCX staining at day 14 after MCAO administration. D–F, Bar graphs showing the percentage of newborn neurons (D), the percentage of neurons with multineurites (E), and total neurite length per newborn neuron with multineurites (F) in the NSCs treated with DETA/NONOate (50 μm) with or without NO scavenger C-PTIO (10 μm) for 24 h at day 2 after differentiation. The cultures were stained 4–5 d after treatment. (n = 3). G, Nitrosylation of total proteins or HDAC2 in NSCs. NSCs were treated with 50 μm DETA/NONOate for the indicated times (G1), with 50 μm GSNO for 4 h (G2), or with 50 μm DETA/NONOate for 4 h (G3) at day 2 after differentiation (n = 4). PC, Positive control, in which the lysate of NSCs was treated with GSNO (200 μm) for 30 min; BSA, biotin-switch assay. H, Immunoblots showing HDAC2 levels in the NSCs treated with 50 μm DETA/NONOate for the indicated times at day 2 after differentiation. (n = 3–5). I, Bar graphs showing HDAC2 activity in the NSCs treated with 50 μm DETA/NONOate for the indicated times (left) or by 50 μm DETA/NONOate with or without 10 μm NO scavenger C-PTIO for 4 h (right) at day 2 after differentiation. n = 3–5. J, K, HDAC2 levels (J; n = 5) and enzymatic activity (K; n = 4) in the solo-cultured or neuron-cocultured NSCs with or without C-PTIO. All cultures were exposed to OGD for 2 h at day 2 after NSC differentiation, and the NSCs were collected 6 h after OGD finish. To measure HDAC2 in NSCs selectively, coculture was performed with neurons in membrane inserts (1 μm pore size) and NSCs in wells. C-PTIO (10 μm) or vehicle was added to the medium of NSCs in wells at the start of OGD exposure. L, M, The percentage of newborn neurons (L) and the percentage of newborn neurons with multineurites (M) in the cocultures of LV-control-shRNA- or LV-HDAC2-shRNA-infected NSCs with GFP+ neurons. The cocultures were subjected to OGD for 2 h at day 2 after differentiation and stained at days 4∼5 after OGD (n = 4). N, O, Bar graphs showing the percentage of newborn neurons (N) and the percentage of newborn neurons with multineurites (O) in the cocultures of AD-HDAC2-flag- or AD-flag-infected NSCs with GFP+ neurons. The cocultures were subjected to OGD for 2 h and treated with 10 μm ZL006 for 26 h beginning at OGD administration, and were stained at days 4∼5 after OGD administration. n = 5. Data are the mean ± SEM. *p < 0.05, **p < 0.01, two-tailed t test for C, ANOVA for others.

Article Snippet: HDAC2 shRNA (m) lentiviral particles (LV-HDAC2 shRNA, Santa Cruz Biotechnology) are recommended for the inhibition of HDAC2 expression in mouse cells.

Techniques: Western Blot, Infection, shRNA, Control, Virus, Staining, Positive Control, Biotin Switch Assay, Activity Assay, Cell Culture, Membrane, Pore Size, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Interaction of nNOS with PSD-95 Negatively Controls Regenerative Repair after Stroke

doi: 10.1523/JNEUROSCI.1305-14.2014

Figure Lengend Snippet: Association of nNOS–PSD-95 in neurons and consequent NO production may negatively regulate NSC fate via HDAC2-mediated histone deacetylation and NeuroD downregulation. A, B, Immunoblots showing acetyl-H4 (A) and NeuroD (B) levels in the NSCs treated by 50 μm DETA/NONOate with or without 10 μm C-PTIO for 8 h at day 2 after differentiation (n = 4). C, D, Immunoblots showing acetyl-H4 (C) and NeuroD (D) levels in the NSCs treated by 50 μm DETA/NONOate with or without 2 nm TSA (an HDAC inhibitor that was added 30 min before DETA/NONOate) for 8 h at day 2 after differentiation (n = 4). E, F, Immunoblots showing acetyl-H4 (E) and NeuroD (F) levels in the NSCs treated with 50 μm DETA/NONOate with or without LV-HDAC2-shRNA infection for 8 h at day 2 after differentiation (n = 4). DETA/NONOate was added at day 6 after LV-HDAC2-shRNA or LV-control-shRNA infection. G, H, Immunoblots (G) and immunofluorescence (H) showing acetylation of histone H4 in the NSCs cocultured with neurons after OGD. Coculture was performed with neurons in membrane inserts (1 μm pore size) and NSCs in wells (n = 4). Data are the mean ± SEM. *p < 0.05, **p < 0.01, ANOVA.

Article Snippet: HDAC2 shRNA (m) lentiviral particles (LV-HDAC2 shRNA, Santa Cruz Biotechnology) are recommended for the inhibition of HDAC2 expression in mouse cells.

Techniques: Western Blot, shRNA, Infection, Control, Immunofluorescence, Membrane, Pore Size