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cmi12021-sup-0001-si.tiff15255K

Fig. S1. Antibody directed against LtxA inhibits LtxA-induced haemolysis. A polyclonal antibody directed against LtxA concentration-dependently decreased LtxA-mediated haemolysis. The antibody was diluted 1/1000, 1/500, 1/100, and 1/50. Values are mean ± SEM, n = 5. The asterisk denotes a statistical difference from control (P < 0.05).

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Fig. S2. LtxA leads to calcium influx in human erythrocytes. Original flow cytometry data from one experiment during control condition (left panel) and 10 min after LtxA is added (10 μg ml−1). Region 1 defines the cell population studied and region 2 defines cells, which are positive to a rise in [Ca2+]i, 2500 cells were investigated per sample.

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Fig. S3. LtxA leads to phosphatidyl serine exposure in human erythrocytes.

A. Six windows displaying original flow cytometry data from one experiment during control conditions, showing the definition of the seven regions described in the table in (B). 10 000 cells were investigated per sample. Region 1 defines EV of the cells investigated, region 2 SS and region 3 numbers of cells positive for annexin V staining from the cells defined in region 1. Region 4 defines shrunken cells (cells with a lower EV and higher SS relative to control) and region 6 swollen cells (cells with a higher EV and lower SS relative to control). Region 5 defines the cells from region 4, which are positive to annexin V staining (= shrunken and PS exposing cells) and region 7 the cells from region 6, which are positive to annexin V staining (= swollen and PS exposing cells).

B. Table with mean values from six experiments during control conditions and after LtxA (1 and 2.5 μg ml−1) and ionomycin (1 μM) were added. All values are mean ± SEM, n = 6. The asterisk denotes a statistical difference from control (P < 0.05).

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Fig. S4. LtxA leads to phosphatidyl serine exposure in human erythrocytes. Human erythrocytes incubated with LtxA (1 μg ml−1) or ionomycin (1 μM, positive control) in combination with FITC-conjugated annexin V in annexin V binding buffer and placed on coverslips. Three DIC images (top panel) and the corresponding fluorescent images (lower panel) from one representative experiment showing a rise in fluorescence when LtxA or ionomycin is present. FITC fluorescence correlated with shrinkage of the cells (indicated by the arrows). The experiment was repeated four times with similar results.

cmi12021-sup-0005-si.tiff17062K

Fig. S5. A. Mechanosensitive cation channels are not involved in LtxA-induced haemolysis. Erythrocytes were incubated for 60 min. Values are normalized to a control without antagonist showing ∼ 50% lysis. All experiments were conducted at 37°C under a constant swirl of 250 r.p.m.

B. Decreasing the concentration of extracellular Na+ does not affect the LtxA-induced haemolysis. Erythrocytes were incubated for 60 min. All experiments were conducted at 37°C under a constant swirl of 250 r.p.m.

C. BBG, a P2X7 antagonist, decreases LtxA-mediated haemolysis. Erythrocytes were incubated for 60 min. Values are normalized to a control without antagonist showing ∼ 50% lysis. All experiments were conducted at 37°C under a constant swirl of 250 r.p.m.

D. PPADS does not affect LtxA insertion into the erythrocyte membrane. Human erythrocytes were incubated for 30 min with the general P2 antagonist PPADS at increasing concentrations. After 30 min unbound PPADS was washed off (1000 g × 3) and the cells were resuspended in HBS containing LtxA (10 μg ml−1) and incubated for 60 min and haemolysis was measured as the optical density at 540 nm of the supernatant. Values are mean ± SEM, n = 5–8. The asterisk denotes a statistical significance from control (P < 0.05).

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Video S1. Time lapse of human erythrocytes on an inverted microscope at 37°C for 60 min. LtxA (10 μg ml−1) was added at time 0. Imaging rate was 0.5 Hz.

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