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. Author manuscript; available in PMC: 2021 Sep 9.
Published in final edited form as: Cell Host Microbe. 2020 Jun 15;28(3):402–410.e5. doi: 10.1016/j.chom.2020.05.012

Figure 2. EMS-induced mutagenesis on CHO cells reveals that a cellular receptor is required for the HBL’s cytolytic activity.

Figure 2.

A. Schematic representation of EMS-induced mutagenesis to isolate HBL-resistant CHO mutant cells. CHO cells (50 million) seeded in ten 10-cm dishes were treated with 0.25 mM EMS for 24 h. Then, the cells were treated with HBL (2.5 nM each component) for 3 h. After toxin removal, the toxin-treated cells were cultured for one week, allowing formation and isolation of HBL-resistant CHO mutant clones.

B. Cytotoxicity of HBL to ten independent (from different dishes) HBL-resistant clones isolated in (A). The cells were incubated with various concentrations of HBL for 2 h, followed by an MTT assay to assess cell viability. Data are represented as mean ± SD.

C. HBL-resistant CHO mutant cells cannot bind HBL. Three representative HBL-resistant clones (CHO-R1, -R2, -R3) were incubated with HBL-B for 1 h, followed by sequential staining with a mouse anti-HBL-B serum and an anti-mouse IgG conjugated with Alexa Fluor 594.