Alexa fluor 488

The M17 and PC12 cells were seeded in 24-well glass slides. After treatment, they were fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature, washed thrice using PBS, permeabilized using 0.1% Triton X-100, blocked using 5% BSA for 1 h, and incubated overnight with primary antibodies at 4°C. The cells were then incubated with Alexa fluor-488 conjugated anti-rabbit antibody (1 : 500) for 1 h at room temperature, and the nuclei were stained with DAPI (Beyotime Biotechnology). Samples were imaged through TSC SP8 confocal microscopy (Leica).

HIECs were seeded onto glass bottom cell culture dishes (801002, NEST, China), and pretreated with or without 0.05 μM DSF for 12 h before 6 Gy radiation. Cells were collected at indicated time points (0, 0.5, 1, 2, 4, 12 and 24 h after radiation), fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with goat serum, and incubated with anti-γH2AX antibody (05636 Merck Millipore, Germany) overnight at 4°C, followed by incubation with Alexa Fluor 488 (A0428, Beyotime, China), Hoechst (C1026, Beyotime, China) counterstaining and observed by confocal microscopy (LSM-700, ZEISS, Germany). The γH2AX-focis were analyzed from the images of IF staining by using ImageJ software 1.8.0 (National Institutes of Health, United States).

Cells were seeded onto cover slips in the six-well plates and incubated for 24 h. Cells were stained with LysoTracker Red at a final concentration of 50 nM. Then, cells were fixed in 4% paraformaldehyde for 20 min, blocked with 5% BSA for 2 h, and incubated with the primary antibody for LC3 (1:150, Abcam, Cambridge, MA, USA, ab63817) or P62/SQSTM1 (1:500, Abcam, Cambridge, MA, USA, ab56416) at 4°C overnight. After that, cells were washed and incubated with a secondary antibody conjugated to Alexa Fluor 488 and Alexa Fluor 555 (Beyotime Institute of Biotechnology, Jiangsu, China). The cells were then counterstained with DAPI (Beyotime Institute of Biotechnology, Jiangsu, China) for 10 min and visualized with a confocal microscope.

The cerebral cortical neurons were plated at a density of 2 × 10⁵ cells on 0.01% poly L-lysine-coated sterile coverslips. After treatment, the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 at room temperature for 20 min, blocked dropwise with 5% bovine serum albumin (BSA), and incubated at 37 °C for 30 min. The fixed cells were incubated with mouse anti-LC3B (1:300) or rabbit anti-LAMP2 (1:100) overnight at 4 °C. The slides were then washed three times with phosphate-buffered saline (PBS) and incubated at room temperature with Alexa Fluor 488 or a cy3-labeled secondary antibody (Beyotime, Shanghai, China) at a 1:200 dilution based on the source of the primary antibody. Finally, the cells were stained with DAPI, observed, and photographed using a laser confocal microscope (Leica, Wetzlar, Germany). The images for the colocalization analysis (colocalization of LC3 with LAMP2) were calculated using the JaCoP plugin in Image J after the thresholding of individual frames. The colocalization analysis was performed on three independent studies with 50 cells per condition in each experiment.

SW1116 cells were treated with CRC exosomes with miR‐1270, miR‐370‐5p, or miR‐140‐5p overexpression, and BeyoClick™ EdU Cell Proliferation Kit assessed the proliferation abilities with Alexa Fluor 488 (C0071S, Beyotime Biotechnology, Shanghai, China). After treating CRC exosomes with different miRNAs overexpression, SW1116 cells with 60% confluence were incubated with Leibovitz's L‐15 medium containing 20 μM EdU reagents for 2 h at 37°C. After removing the medium, SW1116 cells were fixed with 4% paraformaldehyde fix solution (P0099), washed three times with Immunol Staining Blocking Buffer (P0102), and permeabilized by Enhanced Immunostaining Permeabilization Buffer (P0097). All solutions and buffers were purchased from Beyotime Biotechnology (Shanghai, China). In addition, the nuclei were stained with 1X Hoechst 33342 (C0071L‐6, Beyotime, Shanghai, China). EdU‐stained SW1116 cells in all groups were observed and assessed the proliferation ability was assessed by EVOS M5000 fluorescence microscope (Thermo Fisher Scientific, MA, USA).

After treatment and fixation, cardiac fibroblasts were incubated with blocking solution at room temperature. Then, diluted primary anti-α-SMA (1:100), collagen I (1:200), and collagen III (1:200) (Boster Biological Technology, Dublin, CA, USA); ki67 (1:100), PCNA (1:200) (ABclonal Technology, Wuhan, China); and the DRP1, OPA1, or RIPK3 (1:50, Cell Signaling Technology, Danvers, MA, USA) antibody were added and incubated overnight at 4 °C. After washing, the cells were incubated by IgG conjugated with Alexa Fluor 488 or Cy3 (1:500, Beyotime, Shanghai, China) without light at room temperature for 2 h followed by DAPI staining for 15 s. The cells were observed and photographed with a laser confocal microscope. The protein expression, which is considered as the fluorescence intensity, was quantified using ImageJ software.

Cell proliferation was determined using an EdU Proliferation Kit (Beyotime, C0071S, Shanghai, China). Cells were cultured in a 48-well plate for 24 h, then incubated with 50 mM EdU solution for 2 h and fixed in 4% paraformaldehyde. Subsequently, the cells were permeabilized with 0.25% Triton X-100 for 15 min and sequentially stained with Alexa Fluor 488 (Lot:12194) and Hoechst (Beyotime, 33342, Shanghai, China).The EdU-treated cells were then imaged and assessed using an Olympus FSX100 microscope (Olympus, Tokyo, Japan).