4B). In order to clarify the role of the different C/EBP β isoforms in neuronal survival or death, we transfected primary cultures of rat CGNs with plasmids expressing LIP, LAP1, or LAP2, together with a plasmid expressing GFP, and we shifted these differentiated neurons to K5 medium for 24 h in order to induce apoptosis. The transfection efficiency was ~ 20% for all plasmids, which is a relevant percentage for primary neuronal

cultures (Zeitelhofer et al., 2009), and each plasmid was able to express the proteins, as previously demonstrated (Fig. 4A). These cultures were stained with Hoechst for counting, selleck inhibitor in either GFP-positive (transfected) neurons or all neurons, total nuclei and condensed nuclei, indicating apoptotic cells, as shown in Fig. 4C. Quantification of apoptotic nuclei showed that the shift to low potassium induced apoptosis in a significant proportion of GFP-transfected cells. In particular, by using the unpaired, two-tailed Student’s t-test, we observed that, whereas there was a statistically significant difference between the K5-exposed GFP-transfected/LIP-transfected neurons and their controls in

the K25 condition (P < 0.0001 and Z = 5.7590 for both GFP and Midostaurin LIP), there was no significance difference between LAP2-transfected neurons in the K5 condition (P = 0.1637, Z = 1.3926) and LAP1-transfected neurons in the K5 condition (P = 0.0623, Z = 1.8641) and their controls at in the K25 condition. Moreover, in cultures second transfected with both GFP and one of the C/EBP β plasmids, both the LAP1 isoform and the LAP2 isoform almost completely reversed the effect of the apoptotic stimulus, whereas LIP

had no effect, P-values being less than 0.0001 for LAP2-transfected CGNs (Z = 4.9314) and for LAP1-transfected CGNs (Z = 4.0793), whereas the P-value was 0.9116 for LIP-transfected CGNs (Z = 0.1110) as compared with CGNs transfected with only GFP in the K5 condition for 24 h; unpaired, two-tailed Student’s t-test. In addition, the percentage of apoptotic cells in LIP-transfected GGNs in the K5 condition was statistically significant (two-tailed Student’s t-test) as compared with LAP1-transfected CGNs (P < 0.0001, Z = 5.1223) and LAP2-transfected CGNs (P < 0.0001, Z = 5.2794) in the K5 condition (Fig. 4D). In order to confirm the pro-survival effect of LAP2 that we observed in primary neurons, we decided to use DAOY cells, a medulloblastoma cell line showing a similar derivation of CGNs. In fact, medulloblastoma is a pediatric tumor that originates from cerebellar neuron precursor cells (Peña-Altamira et al., 2010). Stable DAOY cell line clones overexpressing LAP2 or LIP were prepared as described in Materials and methods. As shown in Fig. 5A, LAP2 and LIP overexpression was confirmed by western blot analysis, in which the expected molecular masses were observed. Surprisingly, LAP2 overexpression gave rise to an unknown intermediate band.