Supplementary Figure 1. The role of LIMK2 overexpression in cofilin activity, F-actin content and
HMGB1 translocation.
( a-c ) SE reduces pLIMK2 T505 (not S283 or S291+293) level, although it increases total LIMK2 protein expression. Cofilin mRNA/protein expressions and cofilin phosphorylation are unaltered by SE induction. LIMK2 siRNA significantly reduces pLIMK2 T505,
S283 and S291+293 levels following SE. LIMK2 siRNA does not affect cofilin mRNA/protein expressions and cofilin phosphorylation as compared to control siRNA infusion. *p < 0.05 vs. non-SE animals, #p < 0.05 vs. control siRNA (n = 5, respectively). ( d, e ) SE decreases phalloidin (a F-actin marker) signal in CA1 neurons showing swollen dendrites with disassembled microtubules and pyknotic nuclei. LIMK2 siRNA attenuated reduction in phalloidin signal and microtubule disassembly induced by SE. Bar = 12.5 μm. *p < 0.05 vs. non-SE animals, #p < 0.05 vs. control siRNA (n = 5, respectively). ( f, g ) LIMK2 siRNA attenuated HMGB1 translocation from the nuclei to cytoplasm induced by SE. Bar = 12.5 μm. *p < 0.05 vs. non-SE animals, #p < 0.05 vs. control siRNA (n = 5, respectively).
Supplementary Figure 2.
Changes in cyclin D1 and CDK4 expression following SE. ( a-c ) Cyclin D1 and CDK4 mRNA/protein expressions increases following SE. However, active caspase-3 is unaltered following SE. *p < 0.05 vs. non-SE animals, #p < 0.05 vs. 1 day-post SE animals (n = 5, respectively).
( d ) Cyclin D1 and CDK4 immunoreactivities are obviously visible in CA1 pyramidal cells following
SE. Bar = 400 μm. ( e, f ) Cyclin D1 expression in LIMK2 positive neurons is lower than that in
LIMK2 negative neurons. A few cyclin D1 positive neurons show TUNEL signals. Bar = 12.5 μm. *p
< 0.05 vs. LIMK2 negative neuron (n = 5, respectively).
Supplementary Figure 3. Effect of WY 14643 (150 μM) on HMGB1 translocation following SE.
( a, b )
WY 14643 attenuates HMGB1 translocation to the cytoplasm following SE. Bar = 12.5 μm. *p < 0.05 vs. vehicle-treated animals in non-SE group. #p < 0.05 vs. vehicle-treated animals in SE group (n = 5, respectively). ( c ) WY 14643 prevents elongation of mitochondrial length accompanied by inhibition of HMGB1 translocation following SE. Bar = 6.25 μm.
Supplementary Figure 4. Scheme of programmed necrotic neuronal death. p27 Kip1 down-regulation by
1

ROCK induces cyclin D1/CDK4 expressions induced by SE. Cyclin D1/CDK4 complex subsequently increases LIMK2 expression independent of cofilin activity. The up-regulated LIMK2 induces necrotic neuronal death by impairment of DRP1-mediated mitochondrial fission. Inhibition of LIMK2 expression and rescue of DRP1 function attenuate this programmed necrotic neuronal death.
2