Mounting medium

Mesangial cells were grown on coverslips in 6-well plates. Cells were fixed in 4% paraformaldehyde (Pierce) and permeabilized in 0.25% Triton X-100 (Sigma) for 10 min. Cells were washed twice in PBS and blocked in 5% goat serum for 1 h at room temperature, followed by incubation with anti-Smad4 and anti-SUMO2/3 antibodies overnight at 4°C. After washing, cells were incubated with rhodamine and fluorescein isothiocyanate-conjugated secondary antibodies (Bio-Synthesis) for 45 min in the dark. The coverslips were washed and mounted onto slides using mounting medium (Beyotime) and imaged with a DMIRE2 laser scanning confocal microscope (Leica, Germany).

The hippocampus sections were washed using phosphate-buffered saline (PBS : PH 7.4), after being dewaxed by the antigen repair method (EDTA : PH 9.0). After that, they were preincubated in 5% BSA in phosphate buffer for 30 minutes, and then they were incubated overnight using primary antibodies for parkin and p62 (1 : 100; Abcam, Shanghai, China) at 4°C overnight and secondary antibodies (1 : 100; Abcam, Shanghai, China) for 1 h, respectively. Finally, the sections were mounted with mounting medium (containing DAPI; Beyotime, Shanghai, China) and photographed with an epifluorescence microscope (Nikon, Tokyo, Japan).

Granulosa cells were collected and washed with PBS three times at room temperature. They were then placed in PBS containing 0.1% Triton X‐100 at room temperature for 30 minutes. After blocking in PBS‐5% bovine serum albumin (BSA) at 37°C for 1 hour, the granulosa cells were incubated with the special antibodies described above at 4°C overnight. Cells were further incubated with goat anti‐mouse and goat anti‐rabbit at 37°C in the dark followed by staining of nuclei with DAPI for 5 minutes. Finally, all granulosa cells were placed on slides with mounting medium (Beyotime) and gently covered with ethanol‐primed coverslips and kept in the dark until confocal scanning.

RAW264.7 cells were seeded on coverslips and stimulated as indicated, fixed with 4% (v/v) paraformaldehyde, permeabilized with Triton X-100 in PBS, blocked with 3% BSA before incubation with primary antibodies followed by Alexa Flour secondary antibody. Nuclei were stained with DAPI in mounting medium (Beyotime). Fluorescence images were taken with a Leica SP8 confocal laser scanning microscope and processed in Image J software. The antibodies used were rabbit anti-STING (19851-1-AP, Proteintech), anti-iNOS (ab178945, Abcam), and Alexa Flour 488 (ab150077, Abcam).

Liver sections (4 µm) were dewaxed, rehydrated regularly and treated with 3% H₂O₂. The specimens were incubated overnight at 4 °C with anti-𝛼-SMA (1:500, Invitrogen). The cryostat sections of the terminal ileums were incubated with anti-ZO-1 (1:100, Invitrogen) antibodies overnight at 4 °C, followed by incubation with FITC-conjugated goat anti-rabbit secondary antibody (1:100, Beyotime) for 1 h at room temperature. The sections were then washed in PBS, mounted with mounting medium (Beyotime) and photographed under a fluorescence microscope (Eclipse 80i; Nikon, Tokyo, Japan).

Immunofluorescence staining of Ki67 and TUNEL were performed to detect cell proliferation and apoptosis, respectively. Cells cultured on coverslips in 35-mm culture dishes were treated with PPD to ensure a discrimination of individual nuclear foci in immunofluorescence staining. After 24 h incubation, cells were fixed with 4% paraform aldehyde for 20 min at room temperature and permeabilized with 0.1% Triton X-100 (Sigma, Louis, MO, USA) for 10 min at 4 °C. After blocking with 5% Bovine Serum Albumin for 30 min at room temperature, cells were stained with TUNEL reagent (Promega, Madison, WI, USA) for 60 min at room temperature in the dark for in situ apoptosis detection; or incubated with antibody against Ki67 (EMD Millipore, Beijing, China) overnight at 4 °C, followed by incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Thereafter, samples were counterstained with a mounting medium (Beyotime, Shanghai, China) containing Hoechst 33,342. Three random fields of cell samples were inspected with an Olympus BX61 fluorescence microscope (Olympus, Tokyo, Japan), and images were captured on a Zeiss 510 Meta laser scanning confocal microscope (Zeiss, Jena, Germany). Nuclei containing ≥10 immunoreactive foci were scored as positive for TUNEL.