In particular, although we were able to observe LAP1 transient overexpression in DAOY cells (data not shown), after screening of > 40 potential LAP1 stable clones, none of them was positive, suggesting that cells may trigger mechanisms that block LAP1 stable expression. Concerning LAP2 and LIP DAOY stable clones, vitality evaluated with the MTT assay (Fig. 5B) was lower in basal conditions than in EV-transfected stable clones. Moreover, when stable clones
were exposed to 5 μm lactacystin, a well-known and widely used stimulus for inducing neuronal death by blocking the proteasome (Pasquini et al., 2000), control cells transfected with EV were very susceptible to this stimulus, with a decrease in neuronal survival of ~ 40% after 24 h of exposure AZD8055 ic50 for EV clones treated with 5 μm lactacystin vs. untreated clones [one-way anova (F= 15.61, P = 0.0002) followed by the Newman–Keuls comparison test (P < 0.001, mean difference = −34,40, q = 10.08)]. DAOY cells overexpressing LIP showed similar sensitivity to this challenge exposure as untreated LIP-transfected clones (LIP-transfected clones treated with 5 μm lactacystin vs. untreated clones: P < 0.05, mean difference = −13.40,
q = 3.925; same test) and as untreated EV-transfected clones (LIP-tranfected clones Navitoclax treated with 5 μm lactacystin vs. untreated EV-transfected clones: P < 0.001, mean difference = −37.80, q = 11.07; same test), and LIP-transfected untreated clones showed similar sensitivity as EV-transfected untreated clones (LIP-transfected untreated clones vs. EV-transfected untreated clones: P < 0.001, mean difference = −24.40, q = 7.147, same test); however, DAOY cells overexpressing LAP2 showed a statistically significant difference from untreated EV-transfected
clones (LAP2-transfected clones treated with 5 μm lactacystin vs. EV-transfected untreated clones: P < 0.001, mean difference = −22.10, q = 6.473; same test). Moreover, LAP2-transfected cells did not show any significant difference Atazanavir in survival when exposed to lactacystin (LAP2-transfected clones treated with 5 μm lactacystin vs. untreated LAP2-transfected clones: P > 0.05, mean difference = −4.500, same test), further confirming the pro-survival role of the LAP2 C/EBP β isoform in neuronal survival, at least in these treatment conditions. We used rat CGNs, a well-established model of neuronal primary cultures (Contestabile, 2002), in order to study the role of the transcription factor C/EBP β in neuronal survival or death.