PMC

A, GST-AIF67 (10 ng/µl) was incubated with 0–5 U/ml calpain I as indicated, in the absence or presence of 5 mM EGTA. B, Purified mature AIF62 was incubated with 0–5 U/ml calpain I as indicated, in the absence or presence of 5 mM EGTA. Purified AIF57 was used as a molecular marker. Protein was detected using the antibody against AIF.

A, Capn4 PZ/PZ, Capn4 P/P and Capn4 P/P;R MEFs were treated with DMSO, MNNG (500 µM, 15 min) or DPQ (30 µM) + MNNG (500 µM, 15 min). Cell viability was assessed 24 h after treatment by Hoechst 33342 (7 µM, for total nuclei) and propidium iodide (2 µM, for dead cell nuclei) double staining. B, Quantification of MNNG-induced cell death. C, Representative transmission images of Capn4 MEFs 24 h after DMSO or MNNG treatment.

Immunoblots of nuclear and mitochondrial fractions of brain tissue from mice subjected to sham surgery or 90 minutes of middle cerebral artery occlusion and 24 hours of reperfusion. AIF increased in the nuclear fraction after stroke, but the AIF antibody, which recognized recombinant 57, 62, and 67 kDa AIF, recognized only a single AIF close to 62 kDa in the nucleus and mitochondria after sham surgery or stroke. Manganese superoxide dismutase (MnSOD) and histone antibodies were used to assess the integrity of the nuclear and mitochondrial fractions.

Wild-type cortical neurons and neurons transduced by Ad.calpastatin or Ad.GFP were treated with 500 µM NMDA for 5 min or 250 µM NMDA for 2 h. Two hours after the treatment, post-nuclear (PN) and nuclear subcellular (N) fractions were isolated. The integrities of nuclear and postnuclear fractions were monitored by MnSOD and histone immunoreactivity, respectively. A, Representative immunoblots of AIF show nuclear translocation induced by 500 µM NMDA for 5 min. B, Representative immunoblots of AIF nuclear translocation caused by 250 µM NMDA for 2 h. The quantitative values indicated in A & B represent the relative intensity of AIF nuclear translocation normalized to histone. The intensity of AIF in non-transduced cells without NMDA treatment is regarded as 1. Recombinant AIFs, 62 kDa and 57 kDa, serve as molecular markers.

A, Immunoblotting analysis of m-calpain 80 kDa catalytic and 28 or 24 kDa regulatory subunits in Capn4 PZ/PZ (homozygous for the floxed allele), Capn4 P/P (homozygous for Cre-excised allele) and Capn4 P/P;R (Capn4 P/P rescued with lentivirus expressing the small active 24 kDa N-terminally truncated regulatory subunit) MEFs. B, Calpain activity in Capn4 PZ/PZ, Capn4 P/P and Capn4 P/P;R MEFs was determined 2 h after 15 min 500 µM MNNG treatment. ^***p < 0.01. C, Representative immunoblots of MNNG (500 µM, 15 min)-caused AIF nuclear translocation in Capn4 PZ/PZ, Capn4 P/P and Capn4 P/P;R MEFs (upper panel). PN, post-nuclear fraction; N, nuclear fraction. MnSOD was used as a marker of the mitochondrial, postnuclear fraction. Histion and PARP-1 were used as markers of the nuclear fractions. Quantification of AIF nuclear translocation is shown (lower panel). ^***p < 0.001. D, Confocal immunofluorescence images of AIF nuclear translocation in Capn4 PZ/PZ, Capn4 P/P and Capn4 P/P;R MEFs induced by MNNG treatment (500 µM, 15 min). Pink indicates the colocalization of Cy3-stained AIF and DAPI-stained nuclei. Scale bar = 10 µm.

Wild-type cortical neurons (WT) and neurons transduced by Ad.calpastatin or Ad.GFP, were pretreated with or without 30 µM DPQ for 30 min, then exposed to 500 µM NMDA for 5 min (A & B) or 250 µM NMDA for 2 h (C & D). Cell viability was assessed 24 h after treatment by Hoechst 33342 (7 µM, for total nuclei) and propidium iodide (2 µM, for dead cell nuclei) double staining. Representative images of NMDA-induced cell death are shown in A & C, and quantification of NMDA-induced cell death is shown in B & D. Data shown in B and D represent the mean ± SEM of at least three independent experiments. ^***p < 0.001 as compared with cells treated with Controlled Salt Solution (CSS); ^#p < 0.05, ^### p < 0.001 as compared with cells treated with NMDA.

A, Fluorogenic analysis of calpain activity was performed in the whole cell lysate from cells treated with Controlled Salt Solution (CSS) or NMDA under the indicated conditions. The calpain activity in control cells treated with CSS only is regarded as 100%. B, Spectrin immunoblotting analysis (left) was performed on whole cell lysate from cells treated with 500 µM or 250 µM NMDA for the indicated times. Arrows indicate full-length spectrin (240 kDa) and calpain-dependent cleavage products (150 and 145 kDa). The calpain activity is quantified and presented as the percentage of cleavage products in total spectrin (right). n = 8. C, The expression of calpastatin and/or GFP in cortical neurons was assessed by immunoblotting (left) and fluorescence microscopy (right). For immunoblotting, whole cell lysate was prepared from cells transduced with Ad.calpastatin or Ad.GFP for 48 h. D, Calpastatin inhibited calpain activity induced by NMDA (250 µM for 2 h). Data shown in A, B and D represent the mean ± SEM of at least three independent experiments. ^***p < 0.001, ^*p < 0.05 as compared with cells treated with CSS, ^### p < 0.001 as compared with cells treated with NMDA.