G8251

For single‐molecule imaging, a single colony was grown in LB with appropriate antibiotic for 6–8 h the day before the experiment. Eight microliter of culture were diluted in 4 ml of supplemented M9 medium and incubated overnight with the appropriate antibiotic. The next morning the culture was diluted 1/50 and incubated for 2 h with shaking at 37°C. One milliliter of culture was concentrated to 100 μl before labelling the Halotag as described in Banaz et al (2019 (link)) and Cassaro & Uphoff (2022 (link)). Briefly, 5 μl of 50 μM stock solution of TMR dye (Promega G8251) was added to the concentrated culture, mixed vigorously and incubated for 30 min at 25°C. The labelled culture was centrifuged and washed four times with 1 ml supplemented M9 medium to remove the free dye. Cells were then recovered at 37°C with shaking for 30 min and washed again, and 1 μl of concentrated cell suspension was pipetted on an agarose pad (1% agarose in supplemented M9 medium, with or without 100 μM H₂O₂) and sandwiched between two coverslips. Imaging was started as soon as possible after cells were placed on the pads (within ~5 min).

For immunofluorescence, cells were plated on cover glass in a 24-well plate. Four hours after DMSO, T/S/Z or T/S/Z/NSA treatment, cells were fixed and stained as described before [56 (link)]. TMR (G8251, Promega) was supplemented as instructed by manufacturer prior to fixation. All images were taken with Nikon Super-Resolution microscope. For live cell imaging, cells were plated into 35 mm petri dish with glass bottom (P35G-1.5-14-C, MatTek). The following dyes were used according to manufactures’ instructions, Dextran Beads (D1821, ThermoFisher), LysoTracker Red DND-99 (L7528, Thermofisher), LysoTracker Green DND-26 (L7526, Thermofisher), and Sytox Green (S7020, Thermofisher). After adding T/S/Z or D/Z to the plates, cell death was monitored with Nikon A1R microscope.

Localization and tracking of single molecules were done as previously described^{16 (link)}. Briefly, infected HeLa LAMP1-GFP or RAW264.7 cells were labeled with the HaloTag ligand coupled to tetramethylrhodamine (HTL-TMR, Promega G8251) in a concentration of 20 nM for 15 min at 37 °C. After 10 washing steps, the cells were imaged with an imaging medium consisting of Minimal Essential Medium (MEM) with Earle’s salts, without NaHCO₃, without L-Glutamine, without phenol red (Merck D1145) and supplemented with 30 mM HEPES, pH 7.4. TIRF microscopy was performed using an inverted microscope Olympus IX-81, equipped with an incubation chamber maintaining 37 °C and humidity, a motorized 4-line TIRF condenser, a ×150 objective (UAPON 150x TIRF, NA 1.45), a TIRF quadband polychroic mirror (zt405/488/561/640rpc), a 488 nm laser (150 mW, Olympus), and a 561 nm laser (150 mW, Olympus). Localization, as well as tracking of single molecules, were carried out with the help of a self-written user interface in MatLab 2013a^{21 ,46 (link)–50 (link)}.