Fig. 1 ( A ) COX-2 mRNA abundance in hVSMC incubated without (−) and with (+) fetal calf serum (FCS, 2 h) and vehicle (control), cyclosporine A (CsA 100 nmol/L), tacrolimus (Tc 100 nmol/L), rapamycin (Rp 100 nmol/L) and prednisolone (Pd 10 μmol/L); n = 4–5/group; ^*P < 0.05 compared with FCS without drug addition. ( B ) 6-Keto-PGF _1α concentration in the cell-conditioned medium from hVSMC incubated without (−) and with (+) FCS for 20 h with vehicle (ethanol), COX-2 inhibitor NS-398 (NS), cyclosporine A (CsA), tacrolimus (Tc), prednisolone (Pd) and rapamycin (Rp). Values are mean ± SE. ^*P < 0.05 compared with FCS without drug addition. ( C ) COX-2 immunofluorescence labelling of hVSMC cultures without and with FCS and addition of cyclosporine A (CsA 1 μmol/L). Negative control is without primary antibody (Ab). From: Differential effects of immunosuppressive drugs on COX-2 activity in vitro and in kidney transplant patients in vivo Nephrol Dial Transplant. 2009;24(5):1644-1655. doi:10.1093/ndt/gfp004 Nephrol Dial Transplant | © The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.orgOxford University Press

Fig. 8 Effect of immunosuppressives on platelet aggregation in the ex vivo assay. Platelet aggregation ex vivo (% of maximum within 6 min of addition of ADP, 1.0 μmol/L). Values are pooled from determinations before medication at the plasma trough level (0 h) and at the plasma peak concentration (2 h). There were no significant differences between these time points within each of the four groups. Bars show mean. ^*P < 0.001 CsA compared to all other groups by ANOVA followed by Dunnett's multiple comparison test. From: Differential effects of immunosuppressive drugs on COX-2 activity in vitro and in kidney transplant patients in vivo Nephrol Dial Transplant. 2009;24(5):1644-1655. doi:10.1093/ndt/gfp004 Nephrol Dial Transplant | © The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.orgOxford University Press

Fig. 2 ( A ) Amplification of catalytic calcineurin (Cn) subunit Aα, Aβ and Aγ with template cDNA from human cerebral cortex. Negative control (−) is omission of cDNA. Molecular size marker (Mw) is ϕX174DNA/ Hae III digest. ( B ) Amplification of CnAβ (178 bp) and Aγ (108 bp) from cultured, quiescent human aortic smooth muscle cells. Negative control is omission of cDNA (−cDNA) and reverse transcriptase (−RT). Molecular size marker (Mw) is ϕX174DNA/ Hae III digest. ( C ) PCR amplification of cDNA from three separate preparations of human renal arteries for COX-2 (270 bp), CnAβ (178 bp) and CnAγ (108 bp). Negative control (−) is omission of cDNA and positive control (Pc) is amplification of kidney RNA. Molecular size marker (Mw) is ϕX174DNA/ Hae III digest. From: Differential effects of immunosuppressive drugs on COX-2 activity in vitro and in kidney transplant patients in vivo Nephrol Dial Transplant. 2009;24(5):1644-1655. doi:10.1093/ndt/gfp004 Nephrol Dial Transplant | © The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.orgOxford University Press

Fig. 3 ( A ) Plasma concentration of immunosuppressive drugs in the patient groups before ( t =0) and after (2, 4 and 6 h) intake. Values are average ± SE. ^*P < 0.05 by ANOVA followed by Dunnett's post hoc test. Cyclosporine A ( n =11), tacrolimus ( n =8) and rapamycin ( n =9). ( B ) Plasma concentration of prostacyclin metabolite, 6-keto-PGF1α before ( t =0) and after (2, 4 and 6 h) intake of prescribed daily medication in the three patient groups: bars show mean values. ^*P < 0.05 by ANOVA followed by Dunnett's post hoc test. Healthy control group n =11, numbers in other groups as in ( A ). From: Differential effects of immunosuppressive drugs on COX-2 activity in vitro and in kidney transplant patients in vivo Nephrol Dial Transplant. 2009;24(5):1644-1655. doi:10.1093/ndt/gfp004 Nephrol Dial Transplant | © The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.orgOxford University Press

Fig. 4 Effect of E. coli lipopolysaccharide (LPS) on prostacyclin formation in the ex vivo human whole-blood COX-2 assay. ( A ) One millilitre heparin-anticoagulated whole blood from control persons incubated 20 h at 37°C with E. coli -derived 026:B6 LPS (1 and 10 μg/mL) and LPS (10 μg/mL) with the COX-2 selective inhibitor NS-398; n = 4 separate samples. ^*P < 0.05 control versus LPS; ^**P < 0.05 LPS alone versus NS-398 added. ANOVA followed by Dunnett's post hoc test. ( B ) One millilitre heparinized whole blood from control persons incubated 20 h at 37°C without (control) or with E. coli -026:B6 LPS (10 μg/mL) and the synthetic glucocorticoid, dexamethasone (DEXA). WO incubation is a control plasma sample that did not incubate. Values are average ± SEM, n = 7. ^*P < 0.05 LPS versus control; ^**P < 0.05 LPS alone versus DEXA added. ANOVA followed by Dunnett's post hoc test. From: Differential effects of immunosuppressive drugs on COX-2 activity in vitro and in kidney transplant patients in vivo Nephrol Dial Transplant. 2009;24(5):1644-1655. doi:10.1093/ndt/gfp004 Nephrol Dial Transplant | © The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.orgOxford University Press

Fig. 5 Effect of immunosuppressives on COX-2 activity in the whole-blood assay with samples from renal transplant patients and controls. ( A ) One millilitre heparinized whole blood taken before ( t = 0) and after (2, 4 and 6 h) intake of immunosuppressive drugs incubated 20 h at 37°C with LPS (10 μg/mL, E. coli 026:B6). Plasma was separated and analysed for the prostacyclin metabolite 6-keto-PGF _1α : control ( n = 11), cyclosporine A ( n = 11), tacrolimus ( n = 8) and rapamycin ( n = 9). Bars indicate average. ^*P < 0.05. ANOVA followed by Dunnett's post hoc test. ( B ) Left: mean ± SEM plasma concentration of 6-keto-PGF _1α as determined by the whole-blood COX-2 assay in each study group at the peak plasma drug concentration (2 h). Right: whole-blood COX-2 assay with blood from control persons. Cyclosporine A was added directly to blood samples from control persons in a range of concentrations (0.1–100 μmol/L) with LPS (10 μg/mL). Values are average ± SEM, n = 10. From: Differential effects of immunosuppressive drugs on COX-2 activity in vitro and in kidney transplant patients in vivo Nephrol Dial Transplant. 2009;24(5):1644-1655. doi:10.1093/ndt/gfp004 Nephrol Dial Transplant | © The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.orgOxford University Press

Fig. 6 Effect of immunosuppressives on urine excretion of thromboxane A ₂ metabolite, TxB ₂ . ( A ) Excretion of TxB ₂ before ( t = 0) and after (2, 4 and 6 h) intake of immunosuppressives in the three patient groups and in controls receiving no medicine. Bars show mean. Sample sizes were as in Figure 5 . No statistically significant differences were detected by ANOVA. ( B ) Columns depict the mean urine excretion rate of TxB ₂ pooled from the values determined at 0, 2, 4 and 6 h in each of the four groups investigated: control, cyclosporine A, tacrolimus and rapamycin. The sample size in each group is similar to figures above. ^**P < 0.05 CsA versus all other groups, ^*P < 0.05 compared to control. ANOVA followed by Dunnett's post hoc test. From: Differential effects of immunosuppressive drugs on COX-2 activity in vitro and in kidney transplant patients in vivo Nephrol Dial Transplant. 2009;24(5):1644-1655. doi:10.1093/ndt/gfp004 Nephrol Dial Transplant | © The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.orgOxford University Press

Fig. 7 Effect of immunosuppressives on COX-1 activity in the whole-blood assay with samples from renal transplant patients and controls. ( A ) One millilitre whole blood taken before ( t = 0) and after (2, 4 and 6 h) intake of prescribed daily medication incubated 1 h at 37°C in glass vials. Serum was separated and analysed for thromboxane hydrolysis derivative, TxB ₂ : control ( n = 11), cyclosporine A ( n = 11), tacrolimus ( n = 8) and rapamycin ( n = 9). Bars indicate mean. There were no significant differences with time in each group. ( B ) Left: serum concentration of TxB ₂ in each group of patients and controls as determined by the whole blood COX-1 activity assay. TxB ₂ serum concentration values determined in samples taken at 0, 2, 4 and 6 h were pooled in each group and values are mean ± SEM. ^*P < 0.05 compared to control. The Kruskal–Wallis test followed by Dunn's multiple comparison test. Right: thrombocyte count in each of the four study groups. Bars show mean. From: Differential effects of immunosuppressive drugs on COX-2 activity in vitro and in kidney transplant patients in vivo Nephrol Dial Transplant. 2009;24(5):1644-1655. doi:10.1093/ndt/gfp004 Nephrol Dial Transplant | © The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.orgOxford University Press