Cytotoxic effect of Shiga toxin-1 on human proximal tubule cells1 Alisa K. Hughes, Peter K. Stricklett, Donald E. Kohan  Kidney International  Volume 54, Issue 2, Pages 426-437 (August 1998) DOI: 10.1046/j.1523-1755.1998.00015.x Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 1 Cytotoxic effect of Shiga toxin-1 (Stx-1) on human proximal tubular cells (N = 12 each data point). Cells were incubated with Stx-1 for varying times, followed by determination of neutral red uptake. Symbols are: (○) 24 hr; (•) 48 hr; (▵) 72 hr; (▴) 96hr. Kidney International 1998 54, 426-437DOI: (10.1046/j.1523-1755.1998.00015.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 2 Cytotoxic effect of Shiga toxin-1 (Stx-1) on proliferating Vero cells. N = 12 each data point). Cells were incubated with Stx-1 for varying times, followed by determination of neutral red uptake. Symbols are: (○) 24 hr; (•) 48hr; (▵) 72hr; (▴) 96hr. Kidney International 1998 54, 426-437DOI: (10.1046/j.1523-1755.1998.00015.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 3 Competition binding assay of 125 I-Stx-1 to human proximal tubular cells (•) and proliferating Vero cells (○). N = 6 each data point. Approximately 84,000cpm of 125I-Stx-1 were added per well for 24hours at 4°C, cells washed, solubilized, and bound cpm determined. Kidney International 1998 54, 426-437DOI: (10.1046/j.1523-1755.1998.00015.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 4 Total globotriaosyl ceramide (GB3) content in human proximal tubular (Prox. Tub.) and proliferating Vero cells. A standard curve using purified GB3 is shown on the left side of the autoradiogram for comparison. Each experimental lane represents a different cell culture preparation. Details are in the Methods section. Kidney International 1998 54, 426-437DOI: (10.1046/j.1523-1755.1998.00015.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 5 Effect of Shiga toxin-1 (Stx-1) on 3H-leucine uptake by human proximal tubular cells. N = 6 each data point. Cells were incubated with Stx-1 for varying times followed by addition of 1 μCi/ml 3H-leucine for 20minutes. Control 3H-leucine uptake was 2208cpm/mg total cell protein or 253cpm/plate. Symbols are: (○) 24 hr; (•) 48 hr; (▵) 72 hr; (▴) 96hr. Kidney International 1998 54, 426-437DOI: (10.1046/j.1523-1755.1998.00015.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 6 Effect of IL-1 (A), IL-6 (B), TNF (C), LPS (D), or butyrate (E) on Shiga toxin-1 (Stx-1) (10 -9 g/liter) cytotoxicity on human proximal tubular cells. N = 8 each data point. *P < 0.05; **P < 0.01; #P < 0.001 (all P values versus Stx-1 alone). Cells were pre-incubated with inflammatory factors for 24hours prior to exposure to Stx-1 for 72hours and subsequent determination of neutral red uptake. Symbols in A and C are: (▪) 10 U/ml; (□) 100 U/ml; () 1000 U/ml. Symbols in B and E are: (▪) 0.5ng/ml; (□) 2ng/ml; () 5ng/ml. Symbols in D are: (▪) 0.1 μg/ml; (□) 1 μg/ml; () 10 μg/ml. Kidney International 1998 54, 426-437DOI: (10.1046/j.1523-1755.1998.00015.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 7 Effect of 24hours of incubation with 100 U/ml IL-1, 5ng/ml IL-6, 1000 U/ml TNF, 1 μg/ml LPS, or 5mm butyrate on total GB3 content in human proximal tubular cells. N = 6 each data point. *P < 0.025 versus control. Lanes were loaded with an equal weight of total cellular lipids (details are in the Methods section). Kidney International 1998 54, 426-437DOI: (10.1046/j.1523-1755.1998.00015.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 8 Effect of 24hours incubation with 100 U/ml IL-1, 5ng/ml IL-6, 1000 U/ml TNF, 1 μg/ml LPS, or 5mm butyrate on GalT6 activity in human proximal tubular cells. (N = 3 each data point). Control GalT6 activity was 0.38 ± 0.07 densitometry units. Details are in the Methods section. Kidney International 1998 54, 426-437DOI: (10.1046/j.1523-1755.1998.00015.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 9 Effect of 24hours incubation with 100 U/ml IL-1, 5ng/ml IL-6, 1000 U/ml TNF, 1 μg/ml LPS, or 5mm butyrate on binding of 125 I-Stx-1 to human proximal tubular cells. (N = 6 each data point). Details are in the Methods section. Symbols are: (○) control; (•) IL-1; (□) IL-6; (man proximal tubular cells.) TNF; (▵) LPS; (▴) butyrate. Kidney International 1998 54, 426-437DOI: (10.1046/j.1523-1755.1998.00015.x) Copyright © 1998 International Society of Nephrology Terms and Conditions