HBMEC are sensitive to Stxs. (A) Cytotoxic effects of Stx1 or Stx2 (10 ng/ml) on nonconfluent HBMEC were assessed by a neutral red assay at 6, 12, 18, and 24 h after incubation, with the viability displayed at 0 h taken as 100%. (B) Confluent or nonconfluent HBMEC were prepared as described in Materials and Methods and exposed to different concentrations of Stx2. After 18 h, cytotoxicity was measured by a neutral red assay. Cells maintained in medium alone served as the 100% viability control. The error bars show the deviations from three independent experiments.

Flow cytometric analysis of apoptotic markers and electron microscopic analysis. (A) Apoptotic and necrotic cells analyzed using annexin V and PI as markers. Stx2 (10 ng/ml)-exposed HBMEC were harvested at the time points shown and stained by annexin V-EGFP (4 mg/ml) and PI (2.5 mg/ml). The results are depicted as dot plots showing FL1 (488 nm/EGFP) and FL2 (568 nm/PI) channels. (B) Percentages of cells undergoing early apoptosis, late apoptosis, and necrosis were quantitated and represented in the bar graph. The error bars show the deviations among three independent experiments. *, P < 0.05 versus 0-h control (unpaired t test). (C) Decrease of mitochondrial membrane potential (Δψ) induced by Stx2 in HBMEC was detected using JC-1. The results were depicted as dot plots with FL1 (488 nm/monomer JC-1) and FL2 (568 nm/aggregate JC-1) channels. The intact membrane/high Δψ-retaining population and reduced-Δψ population are gated and designated gate 1 and gate 2, respectively. (D) Percentages of gate 1 and gate 2 populations. A shift from gate 1 to gate 2 is the result of a decrease in Δψ. The error bars show the deviations among the three independent experiments. *, P < 0.05 versus 0-h value (Bonferroni t test). (E) Typical chromatin condensations (arrows) were observed in the apoptotic cell at 18 h after incubation with Stx2 (10 ng/ml). Bars, 1 μm.

Activation of caspase-8 and -3 by exogenous rCasp-6 in HBMEC lysate. Active rCasp-6 or the PBS control was added to normal HBMEC extracts, and extracts were incubated for 15 min, 30 min, 45 min, and 60 min. Whole fragments and cleaved fragments of caspase-8 (A) and -3 (C) were detected in the cell extracts containing rCasp-6 by immunoblotting. Addition of PBS to cell extracts did not cause cleavage of caspase-8 (B) or caspase-3 (D).

CHOP knockdown in HBMEC reduces caspase-3 activation and increases viability induced by Stx2. (A) HBMEC were serially transfected with CHOP siRNA-CHOP.1 or -CHOP.2 or with NC-siRNA. Endogenous CHOP mRNA depletion was examined by qRT-PCR analysis. Note that with siRNA-CHOP.1 there was a 55% reduction in CHOP transcript levels compared to that for NC-siRNA (*, P < 0.05, unpaired t test). (B) Caspase-3 cleavage after 18 h of incubation with Stx2 (10 ng/ml) was decreased in the siRNA-CHOP.1-transfected HBMEC compared to that in NC-siRNA-transfected HBMEC. The cleaved caspase-3 was detected by immunoblotting with loading control β-actin. (C) The results additional experiments similar to those for panel B were quantified by densitometry and displayed as percentages of the cleavage in NC-siRNA-transfected cells (100%). Caspase-3 cleavage was statistically decreased in the siRNA-CHOP.1-transfected HBMEC compared to NC-siRNA-transfected HBMEC (*, P < 0.05, unpaired t test). (D) The viability of Stx2-treated HBMEC which were transfected with siRNA-CHOP.1, siRNA-CHOP.2, or NC-siRNA was measured by a neutral red assay. The siRNA-CHOP.1-transfected cells were significantly resistant to the toxin compared to the cell viability of NC-siRNA-transfected cells (P < 0.05, unpaired t test) at doses of Stx2 from 0.1 to 1,000 ng/ml. The error bars show the deviations among the 4 wells in 96-well culture plates. The experiment was repeated three times with similar results.

Detection of the Stx1 receptor glycolipid (Gb₃) in RPTEC, HBMEC, and THP-1 cells in a Stx1 overlay assay. RPTEC, HBMEC, and THP-1 cell neutral glycolipids were prepared from 10⁶ cells and separated by TLC. The relative Gb₃ concentrations were estimated to be 1.6 nmol (RPTEC), 0.4 nmol (HBMEC), and 1.4 nmol (THP-1) based on a standard of Gb₃.

Detection of DNA fragmentation and elements of the caspase cascade induced by Stx2 in HBMEC. (A) Stx2-induced DNA ladder formation and the decrease of the ladder due to caspase inhibitors in HBMEC. Lane 1, control; lane 2, Stx2 (10 ng/ml) alone; lane 3, Stx2 with a general caspase inhibitor; lane 4, caspase-1 inhibitor; lane 5, caspase-2 inhibitor; lane 6, caspase-3 inhibitor; lane 7, caspase-6 inhibitor; lane 8, caspase-8 inhibitor; lane 9, caspase-9 inhibitor. (B) The DNA fragments were quantified by a modification of the diphenylamine method of Burton (5) described in Materials and Methods. The error bars show the deviations among three independent experiments. *, P < 0.05 versus Stx2 alone. (C) Induction of caspase cascade proteins by Stx2 (10 ng/ml) in HBMEC was analyzed by Western blotting. Detected bands of FLIP_L, cleaved caspase-8, cleaved caspase-6, and cleaved caspase-3 as well as loading control, β-actin, are shown. Procaspase-1 was not changed (data not shown). (D) Bands of full-length Bid, cytochrome c, caspase-9, and the loading control, β-actin, are shown. Note that full-length Bid was reduced after 6 h of Stx2 (10 ng/ml) incubation with HBMEC, indicating an increase in active/truncated Bid (tBid). Cytochrome c release from mitochondria and cleavage of caspase-9 occurred following Bid activation. The experiment was repeated twice with similar results.

Validation of CHOP mRNA expression induced by Stx2 in HBMEC. HBMEC were incubated with Stx1 (10 ng/ml), Stx2 (10 ng/ml), Stx1_R170L (10 ng/ml), tunicamycin (1 μg/ml), or etoposide (100 μM) for the times indicated. The amount of CHOP mRNA expression was analyzed by qRT-PCR and depicted as the ratio of mRNA expression to Stx1 mRNA expression at 0 h. The error bars indicate the deviations among three independent experiments. *, P < 0.05 for Stx1 or Stx2 versus Stx1 _R170L or etoposide (unpaired t test).