Fig. 2: Killing by napabucasin depends on ROS. | Nature Communications

Fig. 2: Killing by napabucasin depends on ROS.

From: 2-oxoglutarate:acceptor oxidoreductase-catalyzed redox cycling effectively targets coccoid forms of Helicobacter pylori

Fig. 2

a, b ROS production in H. pylori G27 treated with indicated concentrations of napabucasin (NPB) or antibiotics for 4 h. ROS levels were quantified using a Cell ROS Assay kit (a) and monitored by fluorescent microscopy (b). LVX, levofloxacin; MTZ, metronidazole; CLR, clarithromycin; AMX, amoxicillin. c Schematic representation of reactive oxygen species production and antioxidant defense enzymes in H. pylori. e−, electron, O2• −, superoxide anion; OH•, hydroxyl radical; NO−, nitric oxide; ONOO−, peroxynitrite anion. d Measurement of oxygen consumption rates (OCR) in H. pylori G27 treated with 1× MIC of NPB (0.031 μg/ ml), MTZ (2 μg/ml), AMX (0.063 μg/ml), carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 0.5 μg/ml) or 1% DMSO, using the MitoXpress® Xtra-oxygen probe. Relative OCR was calculated using the slope of the linear portion of fluorescence profiles for each treatment. e Killing efficacy of NPB, MTZ, and AMX under microaerobic and anaerobic conditions. H. pylori G27 cultures were split, diluted, and grown in BHI broth containing 10% FCS treated with 1× MIC of napabucasin, MTZ, and AMX, and incubated either microaerobically in a tri-gas incubator or anaerobically in an anaerobic chamber. Samples were removed, washed, diluted, and plated for colony counts at the indicated time points. f Susceptibility of H. pylori G27 and four mutant strains to NPB and antibiotics. The MICs were determined by broth microdilution assays, as described in Methods. g Viability of H. pylori G27 and four mutant strains treated with the indicated concentrations of NPB. If no colonies were present, calculations were made using the limit of detection (1 × 101 CFU/ml). h ROS were quantified in cells of H. pylori G27 and three mutant strains treated, for 4 h, with 1× MIC of NPB or MTZ. Statistical significance was calculated using a one-way ANOVA with Tukey’s post-hoc test. All data are represented as mean ± SEM (n = 3 biological replicates) except for b, c. Source data are provided as a Source Data file.

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