Volume 55, Issue 2, Pages 546-553 (February 1999) Effect of recombinant human erythropoietin on endothelial cell apoptosis  Raul G. Carlini, Evelyn J. Alonzo, Jose Dominguez, Isaac Blanca, Jose R. Weisinger, Marcos Rothstein, Ezequiel Bellorin-Font  Kidney International  Volume 55, Issue 2, Pages 546-553 (February 1999) DOI: 10.1046/j.1523-1755.1999.00266.x Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 1 Effect of increasing concentrations of recombinant human erythropoietin (rHuEPO) on lipopolysaccharide (LPS)-induced apoptosis. The percentage of apoptotic cells was measured by flow cytometric DNA analysis. Cells were treated with the indicated concentrations of rHuEPO for 24hours in the absence of LPS (▪). Cells were incubated at increasing concentration of rHuEPO (0 to 200 U/ml) for 20hours followed by the addition to the medium of LPS (100ng/ml) during four hours (). Results represent the mean ± sd of three different experiments performed in duplicate. *LPS (100ng/ml) versus rHuEPO (0 U/ml; P < 0.001). **LPS (100ng/ml) versus LPS + rHuEPO (100 U/ml; P < 0.001). ***LPS (100ng/ml) versus LPS + rHuEPO (200 U/ml; P < 0.01). Kidney International 1999 55, 546-553DOI: (10.1046/j.1523-1755.1999.00266.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 2 Flow cytometric analysis of bovine pulmonary artery endothelial cells (BPAECs) treated as follows: serum free medium (SFM) for 24hours (A), only recombinant human erythropoietin (rHuEPO; 100 U/ml) 24hours (B), SFM for 20hours followed by the addition to the medium of lipopolysaccharide (LPS; 100ng/ml) during four hours (C), and cells cultured for 20hours with rHuEPO followed by the addition to the medium of LPS for four hours (D). Results represent the mean ± sd of three different experiments performed in duplicate. Kidney International 1999 55, 546-553DOI: (10.1046/j.1523-1755.1999.00266.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 3 Photomicrograph of BPAECs stained with propidium iodide. (A) Cells cultured for 20hours in serum free medium (SFM) followed by the addition to the medium of lipopolysaccharide (LPS; 100ng/ml) for four hours. (B) Cells cultured with recombinant human erythropoietin (rHuEPO; 100 U/ml) for 20hours followed by the addition to the medium of LPS during four hours. (C) Control cells cultured during 24hours in SFM. Fifty to 100 cells were counted and considered as N = 1. All experiments were performed three times from separate batches of cells. The percentage of apoptotic cells was calculated as follows: the total number of cells with apoptotic nuclei/total number of cells counted × 100. Kidney International 1999 55, 546-553DOI: (10.1046/j.1523-1755.1999.00266.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 4 ELISA quantitation of cellular DNA fragmentation from BPAECs treated as follows: (□) 24hours of incubation in SFM; () 24hours of incubation with rHuEPO 100 U/ml; () 20hours in SFM followed by the addition to the medium of LPS (100ng/ml) during four hours; and () 20hours of incubation with rHuEPO followed by the addition to the medium of LPS for four hours. Results represent the mean ± sd of three different experiments performed in duplicate. LPS (100ng/ml) versus *LPS + rHuEPO (100 U/ml) (P < 0.001). Kidney International 1999 55, 546-553DOI: (10.1046/j.1523-1755.1999.00266.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 5 Representative gel electrophoresis analysis of DNA extracted from BPAECs. Lane 1, molecular weights. Lane 2, cells treated with SFM for 20hours followed by the addition to the medium of LPS (100ng/ml) for four hours. Lane 3, cells incubated for 24hours with only rHuEPO. Lane 4, cells incubated for 24hours with only SFM. Lane 5, cells incubated for 20hours with rHuEPO (100 U/ml) followed by the addition to the medium of LPS during four hours. Similar results were observed in four additional experiments. Kidney International 1999 55, 546-553DOI: (10.1046/j.1523-1755.1999.00266.x) Copyright © 1999 International Society of Nephrology Terms and Conditions