Figure 2. Systematic neuroprotection by systemic application of tacrolimus or p50 expression in cultured RGC-5 cells.
A: Quantitative assessment of the protective effects of p50 expression and tacrolimus against NMDA- or glutamate-induced neurotoxicity.
Cell viability was markedly decreased by the application of glutamate or NMDA. Neuroprotective effects of p50 expression or
tacrolimus were observed.
B: western blot analysis indicates the effects of p50 overexpression or tacrolimus (Tac.) pre-treatment on NF-κBp65 and p50
expression in RGC-5 cells after glutamate treatment; nucleus (Nuc.) and cytosol (Cyt.).
C: Electrophoretic mobility shift assay (EMSA) demonstrates the effects of p50 overexpression or tacrolimus pre-treatment on
NF-κB (p65/p50) activation before and after glutammate treatment. Nuclear extracts were incubated with a double-stranded oligonucleotide
spanning the two B binding sites within the murine bax promoter (B sites). The arrows indicate the position of the protein-DNAcomplexes.
Each lane contains the B sites probe (P) without (lane 1) or with the nuclear extracts prepared from control cells (C) RGC-5
cells (lane 2 and 5), RGC-5 cells transfected with the p50 expression vector (lane 3 and lane 6), and RGC-5 cells pre-treated
with tacrolimus (Tac.; lane 4 and lane 7). Western blot analysis using whole cell extracts also indicates Bax levels under
the culture conditions with p50 overexpression (left panel, lane 10, 12) or tacrolimus pre-treatment (right panel, lane 10,
12).
D: The luciferase reporter vectors containing the wild-type or mutated B fragments of the PMBaxSacI bax promoter (−386 to −1)
or the corresponding emptyluciferase reporter vector (shown in
Appendix 1-Figure 1) were transiently co-transfected with or without the NF-κBp50 expression vector into RGC-5 cells. RGC-5 cells were
pre-treated with tacrolimus for 24 h at 48 h after transfections with the luciferase reporter vectors. Following pre-treatment
with tacrolimus, RGC-5 cells were stimulated by glutamate for 24 h, and then luciferase activities were measured. Values were
normalized to those obtained with the co-transfected pSV-β-galactosidase expression vector. Each assay was performed at least
three times in triplicate. Data are presented as the mean for three independent experiments (*S.D.).
E: western blot analysis indicates the effects of p50 overexpression or tacrolimus (Tac.) pre-treatment on caspase 3 and calcineurin
(CaN) activations in RGC-5 cells after glutamate treatment. Although the glutamate treatment activated caspase 3 and cleaved
CaN, p50 overexpression resulted in a decrease in both active caspase 3 and cleaved CaN expression in RGC-5 cells after glutamate
treatment. The pre-treatment with tacrolimus completely inhibited the glutamate-induced activation of calcineurin, and slightly
decreased the level of activated caspase 3.