Figure legends
Figure 1. Scheme of the in vivo study design.
(Experimental 1) Time course of sample preparation. (Experimental 2)
Evans blue assay. (Experimental 3) Behavior test. GA (50
mg∙kg-1, i.p) was injected immediately after SCI and
then received the same dose of GA at 6 h and 12 h, and then further
treated once a day for 7 d.
Figure 2. GA directly binds to the active site of Jmjd3 and
inhibits Jmjd3 activity. (A) Stereoview of the intermolecular
interactions between the bound GA and the Jmjd3 enzyme residues that
line the catalytic pocket shown as dashed lines. (B) The lysates from
OGD/reperfusion-injured bEnd.3 cells were immunoprecipitated with the
anti-Jmjd3, and immunoblotted with the anti-H3K27me3. GSK-J4 was used as
a positive control. Immunoblotting was analyzed quantitatively (n = 5).
All data represent mean ± SD. *p < 0.05 vs. Jmjd3 only.
Figure 3. GA inhibits the activation and expression of Jmjd3
after SCI. Rats were administered immediately with GA (50
mg∙kg-1) after SCI and spinal cord tissues were
isolated at indicated time after injury. (A) The expression level of
Jmjd3 was determined by RT-PCR (at 6 h and 1 d) and (B) Western blot (at
8 h and 1 d) after SCI and each experiment was analyzed quantitatively
(n = 5). (C) The activity of Jmjd3 was measured by Western blot for
anti-H3K27me3 at 8 h and 1 d after SCI and Western blot was quantified
(n = 5). All data represent mean ± SD. *p < 0.05 vs. vehicle.
(D) Representative fluorescence microscopic photographs showed that
Jmjd3-positive blood vessel endothelial cells are positive for RECA1 at
1 d after injury. Scale bar, 30 μm.
Figure 4. GA inhibits the increase of BSCB permeability by
suppressing MMP-9 expression and activation after SCI. Rat was
administered with GA after SCI and spinal cord tissues from 6 h and 1 d
were processed for RT-PCR (n = 5) and gelatin zymography (n = 5). (A)
RT-PCR for Mmp-2 and -9 and densitometric analysis of
RT-PCR. (B) Gelatin zymography. (C) Densitometric analysis of
zymography. All data represent mean ± SD. *p < 0.05 vs.
vehicle. (D) Representative spinal cord showing Evans blue dye
permeabilized into moderately injured spinal cord and (E) quantification
of the amount of Evans blue at 1 d after injury. Data represent as mean
± SEM. *p < 0.05 vs. vehicle. (F) The expression levels
of TJ molecules were determined by Western blot with anti-ZO-1 (at 1 d)
and anti-occludin (at 5 d) after injury. Western blot was analyzed
quantitatively (n = 5). Data represent mean ± SD. *p <
0.05 vs. vehicle.
Figure 5. GA suppresses Jmjd3-mediated MMP-3 and -9 gene
activation and inhibits TJs loss in OGD-induced bEnd.3 cells. (A) ChIP
assay was performed in bEnd.3 cells subjected to 1 h of OGD/reperfusion
injury using anti-Jmjd3. The occupancy of anti-Jmjd3 at the MMP-3 and
MMP-9 promoter region was calculated via quantitative PCR. (B) Western
blots for Jmjd3 and H3K27me3 was determined in bEnd.3 cells subjected to
OGD/reperfusion injury. (C) Densitometric analysis of Western blot for
Jmjd3 (Left) and H3K27me3 (Right) (n = 5). (D) Western blots for ZO-1
and occludin was determined in bEnd.3 cells subjected to OGD/reperfusion
injury. (E) Densitometric analysis of Western blots for ZO-1 (Left) and
occludin (Right) (n = 5). Data represent mean ± SD. *p< 0.05 vs. +OGD.
Figure 6. GA suppresses Jmjd3-mediated MMP-3 and -9 gene
activation and inhibits TJs loss in OGD-induced bEnd.3 cells. (A)
Schematic drawings showing infiltrated neutrophils and macrophages
positive cells in both the rostral and caudal to the lesion area.
MPO-positive neutrophils (green) at 1 d and ED-1-positive macrophages
(red) at 5 d in the injured spinal cord tissues. (B) Representative
photographs showing MPO-positive neutrophils and ED-1-positive
macrophages in the dorsal column of injured spinal tissues at 1500 and
2000 μm rostral to lesion epicenter. Scale bar, 50 μm. (C) Relative
fluorescent intensity of MPO- and ED-1-positive cells (n = 5). (D)
Western blot (upper) and densitometric analysis (bottom) of ED-1 at 5 d
after injury. To determine the expression of cytokines and chemokines,
RT-PCR and Western blot were performed at indicated time points after
injury (n =5). (E) RT-PCR and (F) densitometric analysis of cytokines
(Il-1β and Tnf-α (at 2 h), Il-6 , Cox-2 andiNos (at 6 h) after injury). (G) RT-PCR and (H) densitometric
analysis of chemokines (MCP-1 , MIP-1α , MIP-1β andMIP-2α (at 2 h), Gro-α (at 6 h) after injury and (I)
Western blot and densitometirc analysis of inflammatory mediators (iNOS
and COX-2 at 1 d after injury). All data represent mean ± SD. *p< 0.05 vs. vehicle.
Figure 7. GA inhibits apoptotic cell death of motor neurons and
oligodendrocytes after SCI. (A) Representative Cresyl violet staining
showing ventral horn of spinal cord at 3 mm rostral from lesion site at
1 d. Scale bar, 50 μm. (B) The spatial pattern of the number of VMN. (C)
Representative TUNEL staining in the GM of the spinal cord at 2 mm
rostral from lesion site at 1 d and (D) in the WM at 7 mm rostral from
lesion site at 5 d. Bottom panels show high-power views (n = 5). Scale
bars, 20 μm. (E) Quantitative analysis of TUNEL-positive cells. (F)
Immunostaining of cleaved (active) caspase-3 in the WM at 5 d after
injury (n = 5). Scale bar, 20 μm. (G) Immunohistochemical analysis of
cleaved caspase-3 and CC1. Double labeling shows that oligodendrocytes
in the WM were positive for cleaved caspse-3 after SCI (arrow). Scale
bar, 30 μm. (H), Quantitative analysis of cleaved caspase-3-positive
cells. (I) Western blot and (J) densitometirc analysis of cleaved
caspase-3 at 1 d and 5 d after injury (n = 5). All data represent mean ±
SD. *p < 0.05 vs. vehicle.
Figure 8. GA improves functional recovery after SCI. (A) BBB
scores of vehicle and GA-treated groups after injury. (B) Grid walk test
of vehicle and GA-treated groups at 35 d after injury. (C) Inclined
plane test of vehicle- and GA-treated groups after injury. (D)
Representative footprints obtained from each group at 35 d after SCI.
All data are presented as mean ± SEM (n = 10). *p <
0.05 vs. vehicle.
Figure 9. GA alleviates axon and myelin loss and decreases
lesion volume after SCI. (A) Representative photographs of
NF200-positive axons in spinal cords. Sections were selected 2 mm
rostral to the lesion site. Note that GA treatment decreased the extent
of axon loss after injury. Scale bars, 20 μm. (B) Quantitative analysis
of NF200-positive axons in ventral (left) and dorsolateral (right)
funiculi showed that the density of spared axons in the GA-treated group
was significantly higher than that in the vehicle. NF200-positive axons
were counted as described under Materials and Methods. (C)
Representative photographs of 5-HT-positive axons in ventral horn areas
in sections 3 mm caudal to the lesion site. Scale bars, 30 μm. (D)
Transverse cryosections (lateral funiculus) selected from 2mm rostral to
the lesion site were processed for Luxol fast blue staining. Note that
the extent of myelin loss was attenuated by GA treatment compared to the
vehicle control. Scale bar, 100 μm. (E) Representative spinal cord
tissues (1.2 mm from the dorsal surface) showing cavitation in the
lesion site and quantitative analysis at 35 d after injury. Scale bar, 1
mm. All data are presented as mean ± SD (n = 5). *p <
0.05 vs. vehicle.