The Journal of Neuroscience, May 17, 2006, ():
Genes Associated with Adult Axon Regeneration Promoted by Olfactory Ensheathing Cells: A New Role for Matrix Metalloproteinase 2
J. Neurosci. Pastrana et al.
26: 5347
Supplemental data
Files in this Data Supplement:
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Supplemental materials
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Supplementary Figure S1. Gelatinase activity is blocked in the presence of specific MMP inhibitors. Gelatinase activity in the co-cultures was monitored by in situ zymography in the presence or absence of 50 μm of the MMP inhibitor GM6001. We added quenched DQ-gelatin to co-cultures of TEG3 OECs and adult rat retinal neurons (RGCs). Co-cultures were maintained for 48 hours and then DQ-gelatin was added and incubated for 4 hours. FITC-fluorescence due to gelatinase activity was drastically reduced in the presence of GM6001. The labelling with an antibody against GAP43 and DAPI is shown. Scale bar, 20 μm.
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Supplementary Figure S2. MMP2 levels are reduced by MMP2-specific RNAi. A, Immunocytochemistry was performed in TEG3 cells co-transfected with the EGFP plasmid together with the MMP2 RNAi-a, MMP2 RNAi-b or the empty pSuper vector as a control. Images showing EGFP positive cells and their corresponding MMP levels are shown. The amount of MMP2 is reduced in EGFP positive cells transfected with the MMP2 RNAi constructs a and b. Cell nuclei were stained with DAPI. Scale bar, 20 μm. B, MMP2 levels were also determined by western-blot in cell extracts of TEG3 cells transfected with MMP2 RNAi-a, MMP2 RNAi-b or the empty pSuper vector, and α-Tubulin levels were analysed as a control in the same membranes. Densitometric quantification of both the upper band corresponding to the MMP2 proform and the lower MMP2 bands permitted us to establish the corresponding total MMP2/α-Tubulin levels. TEG3 cells transfected with MMP2 RNAi-a and b presented a reduction of 40% and 60% in MMP2 protein, respectively.
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Supplementary Figure S3. MMP2 degradation of the perineuronal net increases the amount of adult retinal neurons that regenerate GAP43-positive axons on PLL. Adult retinal tissue was pre-digested with MMP2 (0.5 μg/ml; MMP2 predigestion) and the dissociated cells were then plated on PLL. After 50 hours in culture, cells were analysed by immunocytochemistry using the GAP43 antibody to label regenerating neurons (green) and WFA to visualize perineuronal nets (red; Scale bar, 20 μm). Adult retinal neurons plated on PLL were also cultured in the presence of purified MMP2 protein (0.2 μg/ml; MMP2 addition) for 50 hours in vitro. The number of adult retinal neurons bearing GAP43 positive axons (related to total cell numbers determined with DAPI) under these conditions was quantified and compared to the controls. MMP2 increases the number of adult retinal neurons bearing GAP43 positive axons. Regenerating neurons are devoid of WFA-positive perineuronal nets (see arrow on right panel). A minimum of 200 cells from each condition were counted and histograms show mean values and standard errors from 3 independent cultures.
