The hemolytic activity of RumC1 was evaluated as previously described (53, 56, 57). Briefly, human erythrocytes (obtained from DivBioScience, NL) were pelleted by centrifugation at 800g for 5 min. Cell pellet was then resuspended in sterile PBS and centrifuged at 800g for 5 min. This step was repeated three times, and erythrocytes were lastly resuspended in PBS at a concentration of 8%. One hundred microliters were then added per well of sterile 96-well microplates (Greiner Bio-One) containing 100 μl of PBS with increasing concentrations of RumC1 (final concentrations ranging from 0 to 200 μM) obtained by twofold serial dilutions. Sterile water and Triton X-100 diluted in PBS at 0.1% (v/v) were used as negative and positive controls, respectively. After 1 hour at 37°C, the microplates were centrifuged at 800g for 5 min. One hundred microliters of cell supernatants was collected and transferred to a new 96-well microplate, and OD405nm was measured using a microplate reader (Synergy Mx, BioTek). Hemolysis caused by RumC1 was expressed as the percentage of total hemolysis given by treatment with Triton X-100 at 0.1%. All experiments were done in triplicate.

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