Fig 1.
Identification of gelatinase isoforms from HT1080 cell conditional medium by 1D and 2D zymography.
After incubating with gelatin Sepharose 4B (gelatin 4B) overnight in conditioned medium of HT1080 cells 1D and 2D zymography was performed. Transparent spots (2D) and bands (1D, right side of the gel) revealed proteolytic activity of gelatinases. (A) 2D zymogram showed a 92-kDa proMMP-9 spot with pI value between 3 and 4, and a 65-kDa proMMP-2 spot with pI value between 4 and 5, corresponding to the respective molecular weights of the bands resolved by 1D zymography on the right of the same gel. (B) Conditioned medium of HT1080 cells treated with or without a broad-spectrum MMP inhibitor 1,10 PA were loaded to two IEF dry strips. After IEF separation, these two strips were cut in the pI values ranging from 3 to 6 based on the 2D zymography mapping in Fig 1A, then loaded on the same SDS-PAGE gel for comparison. MMP inhibitor 1,10 PA attenuated gelatinase activity compared to the untreated control.
Fig 2.
2D zymography reveals gelatinase isoforms from HT1080 cell conditioned medium in vitro exposed to APMA.
After incubating with conditioned medium of HT1080 cells, gelatin 4B were exposed to 1 mM APMA, 2 h for MMP-2 activation or 18 h for MMP-9 activation. (A) 1D gelatin zymography revealed gelatinase activity after exposure to APMA. (B, C) Gelatinase isoforms were detected using 2D zymography. Samples with 2-h and 18-h incubation with or without APMA were loaded to IEF dry strips. After IEF separation, strips were cut in the pI values ranging from 3 to 6 and loaded on the same SDS-PAGE gel for comparison, respectively. (D) Enlarged photograph of the 2D zymogram displayed 4 isoforms and multiple fragments of active MMP-2. These zymograms are representative results from 4 independent experiments.
Fig 3.
2D zymography reveals gelatinase isoforms from LPS-stimulated microglial BV-2 cells conditioned medium.
(A) BV-2 cells were treated with 100 or 500 ng/ml endotoxin lipopolysaccharide (LPS) for 16 hr. Proteolytic activity of gelatinases was visualized as a single band of proform of gelatinases (proMMP-2 and proMMP-9) by 1D zymography. (B) In 2D zymograms, proMMP-9 isoforms were visualized as a serious 105-kDa transparent spots with pI values ranging from 3.5 to 7 and proMMP-2 as a 65-kDa single spot with pI value between 4 and 5. These zymograms are representative results from 3 independent experiments.
Fig 4.
2D zymography detects phosphorylation of MMP-9 isoforms from LPS-stimulated microglial BV-2 cells conditioned medium.
BV-2 cells were treated with 100 ng/mL LPS for 16 h. Conditioned medium was enriched by gelatin 4B, incubated with 10 unit alkaline phosphatase (37°C, 1 h), and then analyzed by 2D zymography. Central spot of MMP-9 shifted from pI 5.60 to 5.25 after alkaline phosphatase treatment. These zymograms are representative results from 3 independent experiments.
Fig 5.
2D zymography reveals gelatinase isoforms in mouse brains after focal cerebral ischemia.
Mice were sacrificed 24 h after filament-induced MCAo in mice. (A) 1D zymography revealed increases in proMMP-9 and act.MMP-9 levels in the ischemic cortex compared with the contralateral cortex. Under these experimental conditions, proMMP-2 was not altered. Densitometry analysis of intensity of gelatinolytic bands represented proMMP-9 and act.MMP-9, n = 4, *, p<0.001, comparing ischemic to contralateral cortex by one-tailed, unpaired Student’s t-test; data are expressed as mean values ± SEM. (B) Brain lysate was incubated with gelatin 4B and applied on 2D gels. In contralateral hemispheres, proMMP-9 was identified as a 105-kDa single spot with pI value between 3 and 4, and proMMP-2 as a 65-kDa single spot with pI value between 4 and 5. In ischemic hemispheres, two streaks of pI values ranging from 5.5 to 8 with the molecular weights at 105 and 95 kDa were identified as proMMP-9 and act.MMP-9, respectively. Ischemic brain lysate was applied on the left side of the gel for comparison. These zymograms are representative results from 3 independent experiments.
Fig 6.
Microglial activation and neuronal cell death in ischemic cortex.
Representative photomicrographs of mouse cortical region of brain sections stained with microglia marker Iba-1, NADPH oxidase maker p47phox, and neuron marker MAP-2. Nuclear DNA was counterstained with Hoechst dye. Scale bar, 50 μm.
Fig 7.
2D zymography reveals enzymatic isoforms of gelatinases in TBI mouse brains.
Mice were sacrificed 6 h after CCI-induced TBI. In contralateral hemispheres, proMMP-9 was identified as a 105-kDa single spot with pI value between 3 and 4 and a 105-kDa streak of pI values ranging from 5.5 to 8. In lesioned hemispheres, proMMP-9 was identified as a streak with higher intensity of pI values ranging from 5.5 to 8. Traumatic brain lysate was applied on the left side of the gel for comparison. These zymograms are representative results from 4 independent experiments.
Fig 8.
2D zymography reveals enzymatic isoforms of gelatinases of brain tissues from ZO rats.
(A) Gelatinase activity from the brain tissues of the Zucker lean and obese rats was visualized using 1D gelatin zymography. ProMMP-9, act.MMP-9 and proMMP-2 were identified as bright bands. For the act.MMP-9, ZO vehicle-treated rats showed stronger bands than Zucker lean rats; Linagliptin ameliorates MMP-9 upregulation. (B) Gelatinase isoforms from ZO rats were visualized with 2D gelatin zymography. ProMMP-9 was identified as a 105-kDa single spot, act. MMP-9 as a 95-kDa single spot both with pI values between 3 and 4, as well as proMMP-2 as a 65-kDa single spot with pI value between 4 and 5. Purified gelatinases were applied on 1D zymography on the left side of the same gel for comparison. These zymograms are representative results from 4 independent experiments.