Mitotracker

The IEC-6 cells were seeded at a density of 5 × 10⁴ per well in 24-well culture plates with a cell lamella. After attachment, the cells were cultured with JAC4 for another 24 h. After incubation, the cells were irradiated using the X-ray (12 Gy). After a 24 h incubation, Mito-tracker (Beyotime) was employed to stain the mitochondria for 30 min, and then the IEC-6 cells were fixed and punched. Next, the cells were specifically blocked for 1 h and then treated with cytochrome c mouse antibody (Beyotime) at room temperature for 3 h. Then, the cells were cultured with a fluorescent secondary antibody for another 1 h. Finally, the cells were imaged by a laser scanning confocal microscope (Carl Zeiss).

Cells were grown on chamber slides precoated with fibronectin (Sigma, FC010). The cells were washed 3 times with phosphate buffer saline (PBS) and then fixed with 4% paraformaldehyde for 15 min; after three times washes with PBS, the samples were blocked with 1% BSA in PBS containing 0.1% Triton X-100(PBS-T) and then incubated with primary antibodies at 4 °C overnight. The next day, after incubation with corresponding secondary antibody and Hoechst 33,342 at room temperature for 2 h, the samples were washed in PBS. Cells were loaded with 200 nM Mitotracker (Beyotime, C1035) for 30 min in a humidified incubator with 5% CO₂ at 37 °C. After fixation, permeabilization, and blocking, cells were then incubated with anti-C1qbp overnight at 4 °C. DHE (Vigorous Biotechnology Beijing Co, Ltd, R001) was used to detect intracellular superoxide. DHE (5 μM) was added to cells and incubated at 37 °C for 90 min in the dark. At the end of the incubation time, cells were washed with PBS. Images were captured under the Zeiss LSM800 confocal microscope. Use ImageJ software to analyze cells.

Mitochondrial ROS and MMP were measured by Mito-SOX (40778ES50; Yeasen biotech, Shanghai, China) and Mitotracker (C1048, Beyotime). The NPCs were seeded in the logarithmic growth period on a 6-well plate, cultured at 37 °C overnight under 5% CO2 saturated humidity and treated as the experimental design. The NPCs were stained and fixed according to the manufacturer’s instructions. Finally, the Mito-SOX and the Mitotracker intensity were observed using the LSM.

A total of 1 × 10⁵ cells were seeded in six-well plates containing cell slides. Transfection was performed after 24 h. Before fixation, DMSO was added for 8 h, followed by CQ for 8 h and CCCP for 4 h. DCFH-DA, MitoSOX Red, MitoPY1, and MitoTracker (1:1000, C1032, Beyotime) were added for 10–30 mins. After 24 h, cells were fixed with 4% formaldehyde for 20 mins at room temperature. Subsequently, the cells were washed three times with PBS and incubated with buffer containing 2% BSA and 0.3% Triton X-100 for 1 hr at room temperature. Then, the cells were incubated with the corresponding primary antibody at 4°C for 4 h. After three washes with PBS, the cells were incubated with the secondary antibody for 2 h at room temperature. Following another wash with PBS, the cells were incubated with DAPI (1:1000) for 5 mins at room temperature (Note: Some cells may skip this step and not incubate with DAPI). Finally, the cells were sealed, air-dried overnight, and then photographed using a confocal microscope with a 20× or 60× oil lens.

Mito-Tracker (#C1049B, Beyotime, Shanghai, China) staining: 3 dpf larvae were cultured in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO4) containing 5 µM Mito-Tracker Red (Molecular Probes) for 2 h. They were subsequently washed three times before imaging. To examine the mitochondrial morphology, the larvae were stained with the Mito-Tracker Red and then subjected to tail fin amputation and then fixed with low melting point agarose for confocal imaging.
For the JC-1 (#C2006, Beyotime, Shanghai, China) staining: 3 dpf larvae were cultured in JC-1 working solution (followed by the instructions) for 2 h. They were then washed three times and imaged. The subsequent steps were similar to those used for Mito-Tracker.

For endocytosis analysis, RM-1 cells were pretreated with 50 μM amiloride or 7.5 mM MβCD for 1 h and then incubated with 10 μM CY7-4 for 20 min before observation via CLSM. For mitochondrial colocalization, cells were seeded in confocal dishes (0.5 × 10⁵ cells per dish in 2 mL of RPMI 1640). After incubation overnight, 100 nM Mito Tracker (Beyotime, China) was added to the medium. Thirty minutes later, the medium was replaced with fresh RPMI 1640 containing CY7-4 (10 μM), followed by staining with 50% (v/v) Hoechst 33342 (Beyotime, China) for another 30 min. Free dye was removed by gently washing the cells with PBS.

Mature podocytes were incubated with mitotracker (Beyotime, CN) staining solution for 30 min in the dark at RT and fixed with 4% PFA overnight at 4 °C, then incubated with DAPI staining solution for 5 min in the dark at RT. Images were taken by a Leica sp8 laser scanning confocal microscope.