Figure 3.

ProTα induced cell death mode switch. (a) Time course of the survival activity of cortical neurons throughout serum-free (SFree) and LD cultures. The survival activity of ProTα was evaluated by the WST-8 reduction activity as the percentage relative to the 0 time activity immediately after the start of the cultures using 96-well culture plates precoated with 0 or 25 pmol/cm² ProTα at 2 h before the culture. (b) Transmission EM analysis at 12 h. Necrosis is characterized by membrane destruction and loss of electron density in the cytosol. Apoptotic features of nuclear fragmentation, but no substantial necrotic features, are observed in neurons treated with 25 pmol/cm² ProTα or 20% CM. The CM, which had been preabsorbed with α-ProTα IgG, shows no apoptosis induction. (c) Double staining of LD cultures with PI (red) and annexin V (green), PI (red) and activated caspase-3 IgG (green), and PI (red) and TUNEL (green) after incubation for 3, 12, and 24 h, respectively. (d) Effects of various inhibitors of PKC and PLC on ProTα-induced survival activity. All the inhibitors were used at 1 μM. The survival activity is inhibited by U73122, a PLC inhibitor; GF109203X, a pan-type PKC inhibitor; and Go6976, a PKCα/β inhibitor, but not by U73343, an inactive isomer of U73122; HBDDE, a PKCα/γ inhibitor; or Rottlerin, a PKCα/δ/θ inhibitor. None of the inhibitors had any significant effects alone (not depicted). (e) ProTα induced PKC activation through PLC. The results represent the mean ± SEM from six independent experiments (a, d, and e). *, P < 0.05 versus vehicle; #, P < 0.05 versus ProTα alone.