Propidium iodide dye

Commercial L929 cells were obtained from Chinese Academy of Sciences, Shanghai, China, and were employed to determine the biocompatibility of all the CS-PVA and CS-PVA-US nanofiber mats. The samples were immersed into commercial cell culture medium for 24 h, and the extract-contained media were harvested. L929 cells were seeded in a 96-well plate with a density of 1.0 × 10⁴ cells per well. After 24 h of culture, 100 μL of the as-harvested extract-contained medium was utilized to further culture cells for another 24 h. Then, a CCK-8 assay (Yeasen Biotechnology Co., Ltd., Shanghai, China) was conducted to quantitatively test the cell viability according to the manufacturer’s protocol. Moreover, a live–dead assay was performed to observe the cell viability from the qualitative perspective. Specifically, calcein-AM dye (Yeasen Biotechnology Co., Ltd., Shanghai, China) and propidium iodide dye (Yeasen Biotechnology Co., Ltd., Shanghai, China) were utilized to stain the live and dead cells, respectively. A confocal microscope (CLSM, Zeiss 900 CLSM, Oberkochen, Germany) was utilized to take photos of live–dead staining. The excitation wavelength and emission wavelength of Calcein AM were 490 nm and 515 nm, respectively. The excitation wavelength and emission wavelength of propidium iodide were 490 nm and 630 nm, respectively.

Cells were transfected with the pCI-neo-Serpine1 or pCI-neo plasmid for 48 h. Then, cells were harvested and fixed overnight using 75% ethanol. After incubation with 50 μg/mL propidium iodide dye (Yeasen) and 100 μg/mL RNase A (Yeasen) for 30 min, flow cytometry (BD Biosciences) was performed to quantify the percentage of cells in each cell cycle phase (G0/G1, S, and G2/M).