Cellrox dye

HUVECs were seeded in an MW96 plate at a density of 10,000 cells/well in a final volume of 200 μL. After overnight incubation, a pre-treatment with 0.5 mM hydrogen peroxide (H₂O₂) was conducted for 15 min to induce oxidative stress. After H₂O₂ removal, dyes were added at working concentrations indicated in Table 1 in presence or absence of different doses of HT-C6. Moreover, 1 h later, the time-lapse assay was conducted in the Operetta High Content Imaging System (PerkinElmer, Inc., Waltham, MA, USA) in which photographs were taken every 20 min for 5 h, maintaining normal incubation conditions of temperature and CO₂.
Image analysis during assay was performed with Harmony software version 4.8 (PerkinElmer, Inc., Waltham, MA, USA). Subsequently, segmentation of the different cellular compartments marked by the dyes was performed, fluorescent signal was measured (fluorescence intensity, FI), the background signal of the CellROX dye (Thermo Fisher Scientific, Waltham, MA, USA) was subtracted, and the following processing step was performed: $$\overline{X}FI=\overline{X}{CellROX}_{ROSSignaling}-\overline{X}{CellROX}_{WholeCell}$$
Finally, the images obtained from the Operetta were analyzed and processed using FIJI software (https://imagej.net/contribute/citing) [21 (link)].

Bacterial cryostocks were revived and a single-cell suspension was made to infect peritoneal macrophages at an MOI of 1:1. At 4 h postinfection, cells were washed twice with sterile 1× PBS to terminate M. tuberculosis infection. Murine peritoneal macrophages were then treated with 0.5 μg/mL of WA and were incubated at 37°C for different times for CFU enumeration (60 (link)), immunoblotting, RNA isolation, or flow cytometry. To determine the intracellular ROS, CellROX dye (Thermo Fisher Scientific) was used as per the protocol supplied by manufacturer.