Figure 4
ICP10PK inhibits apoptosis in ΔRR-infected microglia and CM from the infected microglia have neuroprotective activity. (A). EOC2 and EOC20 cells were infected with ΔRR or ΔPK (moi = 5) or mock infected with PBS, and assayed for apoptosis by TUNEL at 24 hrs p.i. Each experiment was done in triplicate and the % staining cells was determined by counting 5 randomly selected fields, (at least 250 cells each, in a 3 mm2 area). Results are expressed as % TUNEL+ cells/total number of cells determined by DAPI staining. The mean TUNEL+ cells ± SD are shown (***p < 0.001 relative to mock). (B). EOC2 and EOC20 cells were mock infected with PBS or infected with ΔRR or ΔPK (moi = 5) and culture supernatants (CM) were collected at 48 hrs p.i. and UV-treated as described in Materials and Methods. Primary hippocampal neurons treated (3 hrs) with NMDA (50 μM) or PBS, were extensively washed with MEM and re-incubated with a mixture (1:1) of Neurobasal medium containing B27 and CM from the infected microglia. They were fixed 24 h later and assayed for cell death by TUNEL. Each experiment was done in triplicate and the % staining cells was determined by counting 5 randomly selected fields, (at least 250 cells each, in a 3 mm2 area). Results are expressed as % TUNEL+ cells/total number of cells determined by DAPI staining. The mean TUNEL+ cells ± SD are shown (**p < 0.01).