A1r storm

As previously described [15 (link)], the cells were washed once with PBS, incubated in DCFH-DA, and diluted 1000 times in serum-free medium for 20 min. Then, the cells were washed three times with serum-free medium and detected via confocal microscopy (A1R+Storm, Nikon).

After incubation with various IMDQ formulations (equivalent to 10 μM IMDQ) for 24 h, BMDMs were incubated with DQ-OVA (10 μg mL⁻¹, Invitrogen) in heat-inactivated DMEM medium for 15 min, washed twice with PBS and incubated at 37 °C for another 15 min. DQ-OVA (488 nm) fluorescence was determined by confocal microscope (A1R-Storm, Nikon) and flow cytometry to assess the lysosomal degradative capacity of macrophages with different phenotypes.

After dewaxing, rehydration, and blocking with BSA, the kidney cortex sections were treated with anti-8-OHdG antibody (Santa Cruz company, sc-66036) overnight at 4 °C. Then, the Alexa Fluor 488–labeled secondary antibody (A-11001, Thermo Fisher Scientific) was used. For detection of co-localization of Tim-3 and LTL/DBA, the primary antibodies were anti-Tim-3 antibody (1:100, ab185703, Abcam), anti-LTL antibody (1:200, FL-1321-2, Vector Laboratories), and anti-DBA antibody (1:100, FL-1031, Vector Laboratories), the secondary antibody was IgG antibody conjugated with Alexa Fluor 594 dye (1:1000, A21207, Thermo Fisher Scientific). After DAPI staining of nuclei, the sections were photographed under a fluorescence microscope (Nikon A1R Storm).

The cells were seeded in glass-bottom dishes and incubated for 24 h at 37 °C, 5% CO₂ atmosphere to allow cell adherence and growth. For real-time imaging, cell nuclei were counterstained with Hoechst 33342 (5 μg mL⁻¹) firstly and washed with PBS. Subsequently, confocal images were captured by a confocal microscope (A1R-Storm, Nikon, Japan) under 60× oil objective lens using 405 nm (nuclei, Hoechst 33342), 488 nm (OFF-ON module, BDP) and 561 nm (always-ON module, Cy3.5) lasers. The DAPI (450/35), FITC (515/30 nm), TRITC (605/75 nm) emission filters were used for Hoechst 33342, BDP and Cy3.5 imaging, respectively. Image acquisition was performed as soon as the addition of the culture medium containing BiRN (100 μg mL⁻¹), followed by real-time imaging for 30 min with a time interval of 1 min between frames. The raw images were processed by NIS-Elements viewer software (Nikon) and the ratiometric images were generated by ImageJ software (NIH).

RKO cells were seeded on coverslips and treated for 72 h with DMSO as a vehicle or the M36 inhibitor. For staining, a MitoTracker^® Red CMXRos probe (M7512, Invitrogen) was diluted at a working concentration of 250 nM in a fresh growth medium, and was prewarmed at 37 °C. The growth medium of the cultures was carefully removed, and the growth medium supplemented with a MitoTracker probe was added. The cells were incubated for 10 min at 37 °C. After staining, the growth medium was removed, and the cells were fixed in a prewarmed solution of 1% paraformaldehyde (PFA) for 5 min at 37 °C. Subsequently, the cells were permeabilized and blocked with 1X PBS, 0.3% Triton X-100, and 10% SFB for 1 h at room temperature. Then, the cells were incubated overnight at 4 °C with mouse anti-p32 (C1QBP) antibody diluted 1:100 in 1X PBS, 0.1% BSA, and 10% SFB. Next, the cells were incubated for 2 h at room temperature with FITC-conjugated goat anti-mouse antibody diluted 1:200 in 1X PBS, 0.1% BSA, and 10% SFB. Subsequently, the nuclei were stained using a solution containing DAPI diluted in PBS 1X with 0.05% Triton X-100. After washing, the coverslips were mounted with the Vectashield anti-fade reagent. Cell fluorescence was examined using a confocal microscope (Nikon A1R+ STORM).

BUMPT cells were treated with cisplatin or/with sTim-3 protein (80 μg/mL) for 18 h. Then, BUMPT cells were stained with MitoSOX Red dye (an indicator of mitochondrial superoxide, 5 μM, M36008, Thermo Fisher Scientific, USA) for 10 min at 37 °C, and then were observed using a confocal microscope (NIKON A1R Storm).