35 mm glass bottom dishes

A 1-mL aliquot of stationary phase YB57 was collected and plated onto 35-mm glass bottom dishes (NEST, USA), washed with PBS and fixed in 4% paraformaldehyde (4 °C) for 15 min. Subsequently, the bacteria were washed three times (5 min per time) with slight shaking, followed by blocking with 200 μL of 5% bovine serum albumin in PBS for 30 min at 37 °C. After blocking, the bacteria were dyed with 20 μmol∙L⁻¹ Hoechst No. 33342 fluorescent dye for 10 min at 37 °C and washed five times with PBS. The mouse-anti-LysGH15 sera (1:500 dilution, 100 μL) and C54S-LysGH15 (produced in our lab, residue C54 was mutated to serine; the C54S mutation in the full-length LysGH15 results in the complete loss of lytic activity) were incubated at 37 °C for 10 min18 (link). Mixtures or single-mouse anti-LysGH15 serum samples were incubated with the dyed YB57 for 30 min at 37 °C19 (link). Subsequently, the treated YB57 was washed three times (5 min per wash) and incubated with 100 μL of TRITC-conjugated goat anti-mouse IgG (1:1,000) (Abbkine, CA, US) for 30 min at 25 °C. After incubation, the cells were washed five times with PBS42 (link). Laser scanning confocal microscopy (LSCM) was used to detect the fluorescence of the treated cells irradiated at different excitation wavelengths.

Changes in the mitochondrial membrane potential (ΔΨm) were measured by JC-1 staining (Solarbio, China). Cells were cultured on 35-mm glass-bottom dishes (NEST, China). Following the indicated treatments, cells were washed with PBS, incubated with 1 mL JC-1 working solution for 30 min at 37°C in the dark, and washed twice with JC-1 staining buffer.
The wavelength at excitation/emission 525/590 nm was used to assess JC-1 aggregates, and at excitation/emission 488/525 nm was used to assess JC-1 monomers under confocal microscopy (SP8, Leica, Germany) as an indicator of ΔΨm, with green emission indicative of relative depolarization.

DSBs were generated with a UVA laser using a pulsed sub-cell illumination system under a 60 × objective lens for live-cell imaging. Cells were seeded on 35-mm glass-bottom dishes (Nest, China), transiently transfected with GFP-Ku80 overnight, visualised 24 h after transfection with a Nikon AX confocal microscope and micro-irradiated with a λ = 365 nm, 16 Hz pulse, 65% energy UVA laser of a Micropoint Ablation System (Andor, USA). Consecutive images were captured at 10 s interval for 10 min.

The Ad-HBmTur-Mito and Ad-LC3-GFP viral vectors (Hanbio, Shanghai, China) were used to directly detect mitophagosome formation by fluorescence confocal imaging. Briefly, cells were seeded on 35 mm glass-bottom dishes (NEST, China), allowed to be infected with virus for 8 h. The culture medium was then exchanged for fresh medium and cells incubated for 48–72 h before the indicated treatments (hypoxia and drug exposure). Treated cells were fixed with 4% paraformaldehyde and counterstained with 4, 6-diamidino-2-phenylindole dilactate (DAPI) (Solarbio, China, C0065). Confocal images of mitophagosomes were acquired using a confocal microscope (SP8 Leica, Germany).