Dapi reagent

The transfected primary cardiomyocytes were inoculated in a 12-well plate, and the cell density was controlled to about 70%. Subsequently, the primary cardiomyocytes were treated differently. After 8 hours, the cardiomyocytes were washed with precooled phosphate buffered saline (PBS) solution 2 times, 5 minutes each; 4% paraformaldehyde was used to fix the cardiomyocytes for 20 minutes; and the cardiomyocytes were incubated with 0.5% Triton X-100 (Sinopharm Chemical Reagent, Shanghai, China) at room temperature for 10 minutes. After washing the cardiomyocytes with PBS 3 times, each well was incubated with TUNEL reagent (Gene, Shanghai, China) at 37°C for 1 hour. After washing the cardiomyocytes with PBS, the nucleus was stained with DAPI reagent, and the positive cells were observed under an inverted fluorescence microscope (Thermo Fisher Scientific, Waltham, MA, USA). ImageJ software was used to exclude false positives and count EdU positive cells.

The penile tissues were soaked in 4% paraformaldehyde for fixation and then transferred to 30% sucrose in PBS for dehydration overnight. The samples were embedded and cut into 5 μm sections before mounting on glass slides. After immersion and blocking for 1 h, the slides were incubated with primary antibodies against α-smooth muscle actin (α-SMA, 1 : 1000; Abcam) and endothelial nitric oxide synthase (eNOS; 1 : 50; Abcam). Next, the slides were washed and incubated with Alexa Fluor-594-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). The nuclei were stained with DAPI reagent (Thermo Fisher, MA, USA) for 5 min. Finally, the slides were examined under a fluorescence microscope (Leica, Heidelberg, Germany).

hiPSC‐CM at day 15 of differentiation were seeded at a density of 100 000 cell cm⁻² in 48‐ and 96‐well plates. 24 h later, cells were treated with EV samples at a dose of 300 particles per cell (time‐point 0 h). 1 × 10⁻⁶ m of EdU was added to each well. Cells were incubated either for 24 or 48 h in a humidified incubator (37 °C, 5% CO2) and counted by trypan blue exclusion assay. Cells were fixed with 4% paraformaldehyde for 20 min at RT, washed three times with DPBS, permeabilized and blocked with blocking buffer (3% bovine serum albumin, 0.05% Triton X‐100) for 30 min at RT. The Click‐iT EdU (ThermoFisher Scientific) reaction was performed according to the manufacturer's instructions to reveal EdU positive nuclei. For immunostaining, corresponding antibodies were diluted at the desired concentration in blocking buffer [cTnT, 1:200, MS‐295‐PI, Thermo Fisher Scientific; phH3: 1:200, Phospho‐Histone H3 (Ser10) (D7N8E) XP(R) rabbit mAb, 53348S, Cell Signaling Technologies] and incubated with cells overnight at 4 °C. Next day, cells were washed three times with DPBS, and incubated with the appropriate secondary antibody diluted in blocking buffer for 1 h. Nuclei were counterstained with DAPI reagent (ThermoFisher Scientific) in a 1:2000 dilution in DPBS.

To investigate the virus inhibition effect, virus titers and fluorescent spots were determined in T-705 treated cells. The N2a cells were seeded in 24-well cell culture plates at 5 × 10⁴ cells/mL overnight. Then, cells were infected with VSV-GFP at MOI of 0.01 at 37 °C for 1 h. After being washed, cells were inoculated with gradient dilutions of T-705 at concentrations of 0.094 to 3 μg/mL at 37 °C for 48 h. The supernatant was collected and determined by virus titration to analyze the differences in virus titers post T-705 treatment.
The changes in fluorescent spots reflect the viral spread efficacy between cells. Similarly, N2a cells were seeded in 12-well cell culture plates at 1×10⁵ cells/mL overnight. Cells were infected with VSV-GFP at MOI of 0.01 at 37 °C for 1 h. Subsequently, cells were washed and fixed in a medium containing 1 × DMEM, 0.7% low-melting-point agarose, 2%FBS, and T-705 at 3 μg/mL at 37 °C for 48 h. Then, the agarose was removed, and the cells were stained by a DAPI reagent (Thermo Fisher, Waltham, MA, USA) to be visualized by a fluorescence microscope.

6 × 10⁴ cells per condition were incubated on poly-L-lysine coated Hendley-Essex 12 well glass microscope slides for 1 h before being fixed in 4% formaldehyde in PBS. The cells were permeabilized in 0.5% Triton-X-100 PBS followed by 2 h of blocking in 5% BSA, 0.2% Triton-X-100 TBS. Primary antibody was applied overnight in a humidified chamber at 4 °C. Appropriate secondary antibody (1:500 dilution) was applied for further 1 h incubation at room temperature after removal of a primary antibody using PBS 0.1% Tween 20. Antifade mountant with DAPI reagent (Thermo Fisher #P36962) was used to seal each sample and images were captured on the Zeiss Axioimager M1 Epifluorescence and Brightfield Microscope. CellProfiler v2.2.0 image analysis software (CellProfiler) was used to quantify IF signals.

Primary fibroblasts were cultured with a diluted EdU reagent (Kaiji, Nanjing, China). The next day, PBS was used to wash the cells, and after fixation, Triton X-100 (Kaiji, Nanjing, China) was used to break the membrane and 5% goat serum was used to seal it. Then, the reaction solution was prepared according to the kit instructions and incubated for half an hour at room temperature. After the cells were rinsed with PBS three times, a DAPI reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to stain the nuclei. Finally, the cells were observed and photographed under an inverted fluorescence microscope.