Figure 3.

NO differentially regulates cultured retinal neuron viability in a cGKII-dependent manner. In a–d, Endogenous NO promotes neuronal death or survival. E6 (a and b) or E8 purified cultures (c and d) were treated in C0 with 100 μM SNAP, 5 μM ODQ, 800 nM KT5823, 25 μM zaprinast, 50 μM 8Br-cGMP or 10 μM YC-1 and quantified in C4. Cell survival, in graphs (a–d), was evaluated by counting of viable cells in purified cultures. Overall cell number in (a): CT=456 cells, n=4; in (b): CT=528 cells, n=4; in (c): CT=360 cells, n=4; in (d): CT=331 cells, n=4. Data represent the mean±S.E.M. (error bars). *P<0.05, **P<0.01, ***P<0.001 in relation to control, one-way ANOVA. In e, Validation of cGKII shRNA-induced cGKII knockdown in E6 and E8 mixed cultures. pLKO=empty vector (transduction control). In f–h, NO regulates retinal cell death or survival via cGKII. Cell death evaluation in mixed cultures by ethidium homodimer-1 (EthD1; purple) labeling (f) or MTT viability assay (g and h) was carried out in C4. Calibration bars=10 μm. EthD1-positive cells in f: pLKO E6=474 cells and pLKO E8=456 cells, n=3. Data represent the mean±S.E.M. (error bars). ^aP<0.05; ^aaaP<0.001 in relation to pLKO in F.2 and F.1, respectively. ^bP<0.05; ^bbbP<0.001 in relation to pLKO+SNAP in F.2 and F.1, respectively. ^ccP<0.05; ^cccP<0.001 in relation to pLKO+SNAP in F.2 and F.1, respectively; one-way ANOVA. *P<0.05; ***P<0.001 in relation to pLKO in h and g, respectively. ^#P<0.05, ^##P<0.01, ^###P<0.001 in relation to pLKO+SNAP in g and h; N=3, one-way ANOVA