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Corresponding author : Chan Young Shin
E-mail: chanyshin@kku.ac.kr
The File includes:
Method
Nitrite quantification
Measurement of cell viability
Supplementary figures
Fig. S1.
Fig. S2.
Supplementary table
Table S1. List of primer sequences used for RT-PCR analysis.

Method
Nitrite quantification
Production of NO was assayed by measuring the levels of nitrite, the stable NO metabolite, in culture medium. Accumulation of nitrite in the medium was determined by colorimetric assay with Griess reagent. The rat primary astrocytes (2.5×10 5 cells/ml) in 24-well plates in 500 μl culture medium were pretreated with different concentrations of PP2 or Bay for 30 min before being induced with LPS (10 ng/ml) for 24 h. In brief,
100 μl of culture media reacted with an equal volume of Griess reagent (part 0.1% N-(1-
Naphthyl)ethylenediamine and part 1% sulfanilamide in 5% H
3
PO
4
) in 96-well plates for 10 min at room temperature in the dark. Nitrite concentrations were determined by using standard solutions of sodium nitrite prepared in cell-culture medium. The absorbance at 540 nm was determined using a microplate reader
(Spectramax 190; Molecular Devices, Palo Alto, CA). Each experiment was performed in triplicate.
Measurement of Cell Viability
Cultured astrocytes were pretreated with different concentrations of PP2 or Bay for 30 min before being induced with LPS (10 ng/ml) for 24 h. Then, cell viability was assessed by MTT assay. MTT is a water-soluble tetrazolium salt that is reduced by metabolically viable cells to a colored, water-insoluble formazan salt. MTT (1 mg/ml) was added to the cell culture medium. After incubating the plates at 37ºC for 2 h in a 5% CO
2 atmosphere, the MTT-containing medium was replaced with dimethylsulfoxide (DMSO). The absorbance was read at 570 nm with a microplate reader (Spectramax 190; Molecular Devices, Palo Alto, CA).

Results
Effect of Bay compound (61-3606) on PA system in LPS-induced rat primary astrocytes
Astrocytes were pre-treated for 30 min with another Src kinase family inhibitor, Bay (1 and 5 µM) and then stimulated with LPS (10 ng/ml) for 24 h. The effects of Bay on tPA/PAI-1 expression were measured by RT-
PCR (Fig. S1a) and Western blot (Fig. S1b). The levels of PAI-1 in culture media and cell extracts were determined using zymography (Fig. S1c and d). As shown in Fig. 2a, LPS alone markedly induced PAI-1 expression by 428.8 ± 44.0% compared to control level.
Fig. S1. The effect of Bay on PAI-1 activity in LPS-stimulated primary rat astrocytes. Primary astrocytes in serum-free DMEM/F12 were pre-treated with Bay (1, 5 µM) 30 min before LPS (10 ng/ml) treatment. (a) The cultured cells were harvested after 24 h and analyzed for tPA , Pai-1 mRNA expression by RT-PCR. Gapdh levels were measured as internal controls. (b) Cell extracts and culture media were collected after 24 h and analyzed for PAI-1 protein expression by Western blot. β-actin levels were measured as loading controls. (c & d)
The culture media and cell extracts were collected 24 h after LPS treatment and analyzed for tPA/PAI-1 activity by casein zymography (for tPA) and one-phase inverse zymography (for PAI-1). The graphs represent the densitometric quantification data of tPA/PAI-1 band intensity. Values are expressed as mean ± SEM. *** p <
0.001 versus control group; # p < 0.05, ## p < 0.01, ### p < 0.001 verus LPS group (n=4).
Inhibition of induced nitric oxide production by SFKs inhibitors
The rat primary astrocytes were pre-treated for 30 min with PP2 (2 and 20 µM) or Bay (1 and 5 µM) and then stimulated with LPS (10 ng/ml) for 24 h. The cell-culture medium was then harvested. The content of nitrite in the medium was measured by Griess reaction. Results showed that LPS stimulation up-regulated the production of NO to 18.6 ± 2.1 µM. Pre-treatment with PP2 (2 and 20 µM) or Bay (1 and 5 µM) effectively decreased the LPS-stimulated production of nitrite in rat primary astrocytes (Fig. S2 a & c).
Effect of SFKs inhibitors on cell viability in LPS-induced rat primary astrocytes
Rat primary astrocytes were pre-treated for 30 min with PP2 (2 and 20 µM) or Bay (1 and 5 µM) and then stimulated with LPS (10 ng/ml) for 24 h. The cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) assay which was conducted in triplicate. The viabilities of the control cells

were used as the control values at 100%. PP2, at concentrations up to 20μM (Fig. S2 b), or Bay, at concentrations up to 5μM (Fig. 2 d), in combination with LPS (10 ng/ml) was not significantly cytotoxic.
Fig. S2. Effect of SFKs inhibitors on the cell viability and nitric oxide production in LPS-stimulated rat primary astrocytes. Primary astrocytes in serum-free DMEM/F12 were pre-treated with PP2 (2, 20 µM) or Bay
(1, 5 µM), 30 min before LPS (10 ng/ml) treatment. (a & c) The culture media were collected 24 h after LPS treatment and analyzed for the assay of nitrite. (b & d) Viability in PP2 or Bay and LPS treated cells were evaluated using the MTT assay. The results are displayed in percentage of control samples. Values are expressed as mean ± SEM. *** p < 0.005 versus control group; ### p < 0.005 versus LPS group (n=3).

Table S1. List of primer sequences used for RT-PCR analysis.
Gene tPA
Pai-1
Forward sequence (5` to 3`)
AGT TGC AGC GAA CCA AGA TG
GCT CCT GGT CAA CCA CCT TA
Src
Fyn
Gapdh
CTC TGA AGC CAG GCA CCA TGT
CCC TTA AGC CAG GCA CCA TG
TCC CTC AAG ATT GTC AGC AA
Reverse sequence (5` to 3`)
TGC CAC GGT AAG TCA CAC CT
CCC CAC AAA ATT CAA GAC CA
CCA AAG GAC CAC ACA TCC GA
CCA AAA GAC CAT ACG TCA GA
AGA TCC ACA ACG GAT ACA TT