N acetyl cysteine (nac)

The intracellular survival assays of pneumococci were performed as reported previously (15 (link), 16 (link)). For the treatment with NAC (Sigma A9165), A549 cells were incubated with 5 mM NAC for 3 h, while Raw 264.7 cells were incubated with 10 mM NAC for 1 h. The NAC treatment was carried out in parallel to the bacterial infection protocol, as described (17 (link)). Apoptosis/necrosis was quantified by flow cytometry using a propidium iodide labeling kit (ThermoFisher). The cell infection scheme was carried out as described in Fig. S7.
To analyze the emergence of FQ-persistent pneumococci, the bacterial-infected A549 and Raw 264.7 cells were cultured in DMEM-1% FBS-6μg/mL levofloxacin. The pneumococci-infected PLB-985 and PLB-985-KO cells were cultured in RPMI 1640/1% SFB/1,3% DMSO/6 μg/mL levofloxacin. Cell cultures were incubated at 37°C and 5% CO₂ at different time points. Cells were lysed by centrifugation for 10 min at 15,000 g and the bacterial pellet was resuspended in BHI. The number of internalized bacteria was quantified after serial dilutions of lysates and plating on blood agar plates. The number of surviving bacteria obtained at t0 was defined as 100% survival in all the cases, and the data obtained at different time points were used to calculate the respective percentages of FQ persisters.

To determine if NAC supplementation would prevent ARHL and cochlear pathology during NAC treatment and after NAC supplementation was discontinued, one group of dwg/dwg mice was supplied ad libitum with NAC (#A7250, Sigma, St. Louis, MO) dissolved in drinking water (10 g/liter) from 3 weeks of age until 6 months of age. Water bottles containing NAC were changed weekly. NAC supplementation was discontinued at 6 months of age, but mice were evaluated again at 9 months of age to determine if the otoprotective effects of the 6-month treatment would be maintained or lost. To determine if NAC supplementation would affect developmental growth, treated and untreated mice were periodically weighed to the nearest 0.1 gram on a lab scale. ABR thresholds were measured in NAC-treated dwg/dwg mice at 3, 6 and 9 months of age and compared to ABR thresholds of untreated dwg/dwg and WT mice of the same age. Afterwards the cochleae were harvested to assess the degree of hair cell loss.

VC, NAC, and the esterified form of ATX were purchased from Sigma-Aldrich (St. Louis, MO, USA). VC, NAC, and ATX were dissolved in dimethyl sulfoxide solution (DMSO, Sigma-Aldrich) to a concentration of 10 mM. Thereafter, 10 mM antioxidant DMSO solution was dissolved in serum-free DMEM media to a final concentration of 1 mM. To determine an appropriate concentration of VC, NAC, and ATX for further experiments, antioxidants were prepared at three concentrations, 1 mM, 100 μM, and 10 μM, and investigated for their free radical scavenging activity and cell survival using diphenyl-1-picrylhydrazyl (DPPH) assay (Sigma-Aldrich) and Cell Counting kit-8 assay kit (CCK-8, Dojindo, Japan). The final concentration of the antioxidants in our study was 10 μM for all three antioxidants based on the criteria for high free radical scavenging activity and low toxicity for cells (Supplementary Data).

NAC (Sigma-Aldrich, St.Louis, MO, USA) was dissolved in distilled water (NAC water, 5.0 g/l), and normal or NAC-containing water was administered orally to rats for periods indicated in figure legends.

To further confirmed the role of oxidative stress on endothelial dysfunction caused by H. pylori infection, the antioxidant NAC was used to treat the mice in vivo and endothelial cells in vitro. NAC is an FDA-approved drug and has been traditionally considered an antioxidant that effectively attenuates ROS production (15 (link)).
Mice in the NAC treatment group received NAC (Sigma-Aldrich, MO, United States, 1 mg/ml in drinking water) 3 days before the first gavage until the end of the experiment (16 (link)). NAC was changed every other day and covered with aluminum foil to avoid exposure to direct light. For the in vitro study, endothelial cells were incubated with 10 mM NAC as described (17 (link)).