Fig. 3. Non-neuronal cells do not mediate PKC-activator-induced neuroprotection. (A) Schematic representation of the neuroprotection paradigm performed in B. (B) SGNs were cultured in the presence or absence of PMA or PDBu (each at 100 nM) on 96-hour SG cultures activated or not by PMA or PDBu for 30 minutes (see conditions presented in A). The number of viable neurons in the new 24-hour cultures as a percentage of neurons in the untreated control condition (a) was determined (n=6; ^***P<0.001). (C) SG cells were cultured in medium containing BrdU and either vehicle or one of the tested compounds (100 nM PMA, 10% FBS, 10 µM AraC). After 15 or 24 hours, cells were fixed and processed for immunocytochemistry and propidium iodide staining to identify nuclei. The proliferative level in culture was evaluated as the number of BrdU-positive cells per total cell number in the culture. Each determination was performed in duplicate, and repeated in three independent experiments. The mean value is shown (n=6; ns, not significant; ^**P<0.01; ^***P<0.001). (D) SG cells were cultured for 24 hours in the presence or absence of 100 nM PMA and specific anti-proliferative agents, i.e. AraC (10 µM) and FUDR (20 µM). Average numbers of TUJ1-positive SGNs under different experimental conditions were counted, as previously described in Fig. 2A (n=9; ^***P<0.001). (E) AraC was used to block proliferation in culture of SGNs in the presence or absence of 100 nM PMA and, after 72 hours, cells were fixed and neuronal viability was expressed as the percentage of neurons present in the untreated control condition (n=6; ns, not significant; ^***P<0.001).