1. Signal transduction of β2m-induced expression of VCAM-1 and COX-2 in synovial fibroblasts 
Neal X. Chen, Kalisha D. O'Neill, Toshimitsu Niwa, Sharon M. Moe 
Kidney International 
Volume 61, Issue 2, Pages (February 2002)
DOI: /j x
Copyright © 2002 International Society of Nephrology Terms and Conditions

2. Figure 1
β2-microglobulin (β2m) modulates intracellular calcium levels in synovial fibroblast-like cells (SFLs). Cells were loaded with 3 μmol/L fura 2-AM, a fluorescent intracellular calcium ([Ca2+]i) chelator and baseline [Ca2+]i level determined for one minute. Then β2m (10, 25 or 50 μg/mL) or bovine serum albumin (BSA 25μg/mL) was added to the cells and the [Ca2+]i response (y-axis) was calculated by determining the difference between baseline and peak calcium levels. (A) Representative experiment with Ca2+ imaging illustrating response of SFLs to β2m (25 μg/mL). The peak [Ca2+]i response occurred within 40 seconds after addition of β2m (arrow). (B) β2m induced a dose dependent increase in [Ca2+]i concentrations, whereas BSA did not. #Different from β2m (10 μg/mL); *different from cells treated with BSA, P < (C) Influence of various inhibitors in β2m-induced [Ca2+]i response in SFLs. Cells were pretreated with U73122 (PLC inhibitor, 5 μmol/L), 2-APB (IP3 inhibitor, 75 μmol/L) or staurosporin (PKC inhibitor, 50nmol/L) for 30 minutes before the addition of β2m (25 μg/mL) and the [Ca2+]i response was measured. U73122 and 2-APB completely blocked β2m-induced increase in [Ca2+]i, whereas staurosporin had no effect (*different from cells treated with β2m alone, P < 0.05).
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

3. Figure 2
Roles of intracellular calcium ([Ca2+]i) and phosphatidylinositol 3 (PI-3) kinase in β2-microglobulin (β2m)-induced expression of vascular cell adhesion molecule in synovial fibroblasts (SFLs). Cells treated with β2m for 24 hours had increased expression of VCAM-1 (A, B). Cells were also treated with various blockers in the absence or presence of β2m (25 μg/mL) for 24 hours: 10 μmol/L BAPTA-AM (Ca2+ chelator, C, F); 5 μmol/L U73122 (PLC inhibitor, D, G); 5 μmol/L U73343 (negative control for PLC inhibitor, E, H); 75 μmol/L 2-APB (IP3 inhibitor, I); 50nmol/L staurosporin (PKC inhibitor, J, M); 10 μmol/L wortmannin or LY (PI-3 kinase inhibitor, K, N, L, O). Images were recorded on Digital camera and processed on a MetaMorph Imaging System (×20). The results demonstrate that [Ca2+]i, PLC and PI-3 kinase, but not PKC, are essential for β2m-induced VCAM-1 expression.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

4. Figure 3
Roles of [Ca2+]i and PI-3 kinase in β2m-induced expression of COX-2 in SFLs. Cells treated with β2m for 24 hours had increased expression of COX-2 (A, B). Cells also were treated with various blockers in the absence or presence of β2m (25 μg/mL) for 24 hours: 10 μmol/L BAPTA-AM (Ca2+ chelator, C, F); 5 μmol/L U73122 (PLC inhibitor, D, G); 5 μmol/L U73343 (negative control for PLC inhibitor, E, H); 10 μmol/L indomethacin (COX inhibitor, I); 50nmol/L staurosporin (PKC inhibitor, J, M); 10 μmol/L wortmannin or LY (PI-3 kinase inhibitor, K, N, L, O). Images were recorded on Digital camera and processed on a MetaMorph Imaging System (×20). The results demonstrate that intracellular calcium, PLC and PI-3 kinase, but not PKC, are essential for β2m-induced COX-2 expression.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

5. Figure 4
Dependence of β2m-induced expression of VCAM-1 on actin cytoskeleton reorganization in SFLs. Cells were treated with or without β2m (25 μg/mL) for 24 hours and stained for F-actin (A, C) and VCAM-1 (B, D). SFLs were also treated with 1 μmol/L cytochalasin D or latrunculin A, which disrupt actin cytoskeleton reorganization, in the absence (E, F, I, J) or presence of β2m (G, H, K, L) for 24 hours and stained for F-actin and VCAM-1. Images were recorded on a Digital camera and processed on a MetaMorph Imaging System (×20). β2m-induced VCAM-1 expression was attenuated by these inhibitors of the actin cytoskeleton.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

6. Figure 5
Dependence of β2m-induced expression of COX-2 on actin cytoskeleton reorganization in SFLs. Cells were treated with or without β2m (25 μg/mL) for 24 hours and stained for F-actin (A, C) and COX-2 (B, D). SFLs also were treated with 1 μmol/L cytochalasin D or latrunculin A, which disrupt actin cytoskeleton reorganization, in the absence (E, F, I, J) or presence of β2m (G, H, K, L) for 24 hours and stained for F-actin and COX-2. Images were recorded on a digital camera and processed on a MetaMorph Imaging System (×20). β2m-induced COX-2 expression was attenuated by these inhibitors of the actin cytoskeleton.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

7. Figure 6
Western blot analysis of β2m-induced VCAM-1 and COX-2 expression in synovial fibroblasts in the presence of various cell signaling inhibitors. Cells were treated with β2m in the presence of 10 μmol/L BAPTA, 5 μmol/L U73122, 5 μmol/L U73343, 1 μmol/L cytochalasin D, 50nmol/L staurosporin, 10 μmol/L wortmannin, or TNF-α (100 ng/mL, positive control) for 24 hours. Cells were lysed and Western blotting was performed with VCAM-1 or COX-2 antibody using equal loading of total protein concentration of 20 μg/lane. The band intensity was analyzed by scanning densitometry. (A) COX-2 expression in synovial fibroblasts. (B) VCAM-1 expression in synovial fibroblasts. (C) Mean densitometry analysis of COX-2 (□) and VCAM-1 (▪) expression in SFLs combined from three separate experiments, with the conditions correlating to those in panels A and B. Data are shown as mean ± SEM from three experiments. Groups with # and * are different from controls (P < 0.05).
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions