Isolectin B4
Iba1
OPL
OPL
Isolectin B4
Iba1
OPL
GCL
GCL GCL
Normal
Diabetic
H&E
Figure S1.
Micorglial cells clustered around perivascular regions labeled with isolectine B4 in diabetes. Red
color represents microglia (Iba1) and green color represents vascular region (isolectine B4). OPL,
outer plexiform layer; GCL, ganglion cell layer. H & E, hematoxylin and eosin stain.
A. Normal
B. Diabetic
OPL
C.
D.
Iba1
GCL
Iba1
Figure S2.
Microglia in the retina become activated during diabetes. Retinas from control and diabetic rats
were processed for Iba-1 (red) 8 weeks after diabetes was induced. Microglia in the control retinas
possessed thin processes characteristic of ramified microglia (A and C), while in the retinas of
diabetic rats appeared hypertrophic indicative of an activated phenotype (B and D). C and D
represent fivefold enlargements of sections of A and B.
TUNEL
PI
Merge
Bright field
A
B
Figure S3.
Apoptosis in microglia cells after activation with AGA 500 ug/ml. A) TUNEL-positive control obtained by
using nuclease and apoptosis signals were observed in almost all nuclei. B) Microglial cells treated with
AGA for 4 hours and no apoptosis signal was observed. Red images represent nuclear staining (PI;
Propedium iodide) and green images represent positive TUNEL staining.
TNF-α Produced,% of Control
p< 0.001
-
0
+
Normal Control
p< 0.001
1
10
+
+
20 µM SB202190
+
AGA
Figure S4A.
Dose-dependent inhibition of AGA-induced TNF-α release in retinal microglial cells by inhibitor for P38(SB202190).
Cells were treated with 500 μg/mL of AGA for 4 h in the presence of indicated concentrations of the inhibitor. TNF-α
levels were compared to the vehicle-treated control. Data shown are the mean + SD of three experiments.
Cell viability (%)
100
90
80
70
60
50
40
30
20
10
0
0
1
10
Figure S4B.
Effect of SB202190 on cell viability, as determined by trypan blue exclusion test.
20
µM SB202190
P<0.001
40000
Relative DCF
fluorescence/mg protein
N.S
30000
DMSO
20000
SB203580
SB202190
10000
0
BSA
AGA
Figure 4SC. P38 inhibitors do not affect the ROS generation.
Retinal microglial cells loaded with 2′,7′-dichlorodihydrofluorescein diacetate were pre-treated with P38 inhibitors
or DMSO, then treated with AGA, and the fluorescence of DCF was measured at 15 min. ROS formation was
expressed as changes in DCF fluorescence/mg protein. Data shown are the mean + SD of 3 experiments.