Journal: Journal of the American Society of Nephrology

Article Title: Verotoxin (Shiga Toxin) Sensitizes Renal Epithelial Cells to Increased Heme Toxicity: Possible Implications for the Hemolytic Uremic Syndrome

doi: 10.1097/01.asn.0000138547.51867.43

Figure Lengend Snippet: Figure 1. Hemin- and verotoxin (VT)-induced cytotoxicity of ACHN cells. (A) Cell monolayers were treated with vehicle, hemin (100 or 200 M), or VT1 for the indicated time intervals. Images were acquired by phase-contrast microscopy (original magnification, 1:10). (B and C) Monolayers were treated with hemin (B) or VT1 (C) as indicated. Residual cells were stained with crystal violet and monitored by spectrophotometry as described in Materials and Methods. Data points are means and SD of triplicate wells. Where error bars are not present, the SD was too small to appear. Figures are representative of at least three independent experiments.

Article Snippet: Human renal carcinoma–derived tubular epithelial (ACHN) cells were obtained from the American Tissue Culture Collection (ATCC CRL-1611) and propagated in MEM with Earle salts and L-glutamine, supplemented with penicillin-streptomycin and 10% FBS.

Techniques: Microscopy, Staining, Spectrophotometry

Journal: Journal of the American Society of Nephrology

Article Title: Verotoxin (Shiga Toxin) Sensitizes Renal Epithelial Cells to Increased Heme Toxicity: Possible Implications for the Hemolytic Uremic Syndrome

doi: 10.1097/01.asn.0000138547.51867.43

Figure Lengend Snippet: Figure 2. Hemin- and verotoxin (VT)-induced cell death. (A) ACHN cell monolayers were treated with vehicle (NaOH), hemin (100 M, for 16 and 28 h), VT2 (5 nM, for 48 h), or TNF- (10 ng/ml) plus cycloheximide (10 g/ml) (1 h), as apoptosis control. Floating cells were collected, fixed, and stained with DAPI as described in Materials and Methods. The number of floating cells from the control monolayers was too low to be evaluated, and the residual cells were fixed and stained in situ. Arrows point at examples of apoptotic cells, defined by nuclear segmentation in the DAPI-stained preparations. (B) ACHN monolayers were treated with vehicle (Veh), VT2 (1 nM, 3 d), or hemin (200 M, 24 h). Total cellular protein extracts were subjected to Western blot analysis with anti-poly(ADP-ribose) polymerase (PARP) antibody. Gels were stained for proteins and photographed for transfer as loading control.

Article Snippet: Human renal carcinoma–derived tubular epithelial (ACHN) cells were obtained from the American Tissue Culture Collection (ATCC CRL-1611) and propagated in MEM with Earle salts and L-glutamine, supplemented with penicillin-streptomycin and 10% FBS.

Techniques: Control, Staining, In Situ, Western Blot

Journal: Journal of the American Society of Nephrology

Article Title: Verotoxin (Shiga Toxin) Sensitizes Renal Epithelial Cells to Increased Heme Toxicity: Possible Implications for the Hemolytic Uremic Syndrome

doi: 10.1097/01.asn.0000138547.51867.43

Figure Lengend Snippet: Figure 3. Combined effects of verotoxin and hemin. (A) ACHN cell monolayers were treated with vehicle or VT1 10 pM for 18 h, followed by the addition of 100 or 200 M hemin (or vehicle) for another 12 h. Additional dishes were treated with cycloheximide (CHX) 10 g/ml for 30 min instead of VT. Monolayers were photographed in situ with an inverted Zeiss microscope with a 10 objective. (B) ACHN monolayers were treated with diluent or VT1 (1 or 10 pM) for 18 h in 96-well dishes, followed by vehicle or hemin (100 or 200 M) for another 6 or 48 h. Residual cells were quantitated by crystal violet staining. Shown are means and SD of triplicate determinations. * P 0.05; ** P 0.01; *** P 0.005. Results represent at least three similar experiments. (C) Human microvascular endothelial cell (HMVEC) monolayers were incubated with 0.1 pM VT2 (top), or sequentially with VT2 and hemin (50 M) after 18 h (bottom). Hemin failed to induce microscopically discernible monolayer changes at the used concentration when added alone (data not shown). Images were acquired via time-lapse video microscopy. The number of hours elapsed from the addition of VT and of hemin is indicated. For comparison, the cytotoxic effect of a 100-fold higher concentration of VT2 on HMVEC is shown on the right.

Article Snippet: Human renal carcinoma–derived tubular epithelial (ACHN) cells were obtained from the American Tissue Culture Collection (ATCC CRL-1611) and propagated in MEM with Earle salts and L-glutamine, supplemented with penicillin-streptomycin and 10% FBS.

Techniques: In Situ, Microscopy, Staining, Incubation, Concentration Assay, Comparison

Journal: Journal of the American Society of Nephrology

Article Title: Verotoxin (Shiga Toxin) Sensitizes Renal Epithelial Cells to Increased Heme Toxicity: Possible Implications for the Hemolytic Uremic Syndrome

doi: 10.1097/01.asn.0000138547.51867.43

Figure Lengend Snippet: Figure 4. Induction of heme oxygenase-1 (HO-1) by cadmium chlo- ride (CdCl2) in ACHN cells. Confluent monolayers were treated (A) for 6 h with the indicated CdCl2 concentrations, or (B) with 50 M CdCl2 for the indicated time intervals. Total cellular extracts were resolved by SDS-PAGE, blotted, and developed with a polyclonal antibody to HO-1. Gels were stained after transfer to assess relative protein loading as described in Materials and Methods (A). Alterna- tively, blots were stripped and reprobed with a monoclonal antibody to -actin as loading control (B).

Article Snippet: Human renal carcinoma–derived tubular epithelial (ACHN) cells were obtained from the American Tissue Culture Collection (ATCC CRL-1611) and propagated in MEM with Earle salts and L-glutamine, supplemented with penicillin-streptomycin and 10% FBS.

Techniques: SDS Page, Staining, Control

Journal: Journal of the American Society of Nephrology

Article Title: Verotoxin (Shiga Toxin) Sensitizes Renal Epithelial Cells to Increased Heme Toxicity: Possible Implications for the Hemolytic Uremic Syndrome

doi: 10.1097/01.asn.0000138547.51867.43

Figure Lengend Snippet: Figure 5. Verotoxin (VT) abrogates induction of HO-1 protein ex- pression in ACHN cells. Monolayers were sequentially treated with vehicle ( CdCl2 (50 M), VT2 (10 or 100 pM), or hemin (200 M) for the indicated time intervals. Time 0 h corresponds to the addition of VT2. Total cellular protein was extracted at time X. Blots were developed with a polyclonal antibody to HO-1, stripped, and reprobed with a monoclonal antibody to -actin as loading control.

Article Snippet: Human renal carcinoma–derived tubular epithelial (ACHN) cells were obtained from the American Tissue Culture Collection (ATCC CRL-1611) and propagated in MEM with Earle salts and L-glutamine, supplemented with penicillin-streptomycin and 10% FBS.

Techniques: Control

Journal: Journal of the American Society of Nephrology

Article Title: Verotoxin (Shiga Toxin) Sensitizes Renal Epithelial Cells to Increased Heme Toxicity: Possible Implications for the Hemolytic Uremic Syndrome

doi: 10.1097/01.asn.0000138547.51867.43

Figure Lengend Snippet: Figure 6. Cadmium chloride blunts hemin toxicity in verotoxin (VT)-treated ACHN cells. (A) Monolayers were treated as indicated with vehicle, or VT2 1 nM for 24 h, or CdCl2 50 M for 6 h, followed by 200 M hemin for 24 h, or hemin 200 M alone for 24 h. Monolayers were photographed with an inverted microscope, 10 objective. (B) Monolayers in 96-well format were sequentially treated with vehicle or 50 M CdCl2 for 6 h, 0.001 nM VT2 (or diluent) for 9 h, and 200 M hemin (or vehicle) for 24 h. (C) Cells were treated as under (B), except that the VT2 concentrations varied between 0.01 and 1 nM, and hemin treatment was prolonged to 48 h. Residual cells were quantitated by crystal violet staining. Shown are the means SD of triplicate wells, normalized for vehicle-treated controls. * P 0.05; ** P 0.01; *** P 0.005. ¶ P 0.05 compared with hemin, and P 0.005 with VT2 alone (B). # P 0.05 compared with VT2 alone at the corresponding concentrations (C). Data represent at least three independent experiments.

Article Snippet: Human renal carcinoma–derived tubular epithelial (ACHN) cells were obtained from the American Tissue Culture Collection (ATCC CRL-1611) and propagated in MEM with Earle salts and L-glutamine, supplemented with penicillin-streptomycin and 10% FBS.

Techniques: Inverted Microscopy, Staining