Beyoclick edu 594

HA-VSMCs were seeded into a 6-well plate and then transfected or cotransfected with miR-335-5p mimic or NC mimic, SP1/pcDNA3.1 or pcDNA3.1 and SP1/pGPH1 or pGPH1 using Lipofectamine 2000 according to the manufacturer’s protocol for 24 h. The cells were then treated or not treated with PDGF-BB (20 ng/mL; PeproTech) for 24 h at 37°C and then incubated with BeyoClick™ EdU-594 (C0078S; Beyotime) diluted at 1:500 for 2 h at 37°C. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 min, subjected to three 5-min washes with PBS and incubated with PBST (PBS containing 0.3% Triton X-100) for 15 min at room temperature. The cells were then incubated with 500 μL of Click Additive Solution in the dark for 30 min at room temperature and then subject to three 5-min washes with PBST at room temperature. The cell nuclei were stained with DAPI (C1005; Beyotime) and the cells were observed under a TE2000-U fluorescence microscope (Nikon, Tokyo, Japan). The EdU-positive cells in 6 visual fields were detected and counted.

After transfection with indicated constructs, cells were seeded into 24-well plates (0.5 × 10⁵ cells/well) and cultured for 24–72 h. Cells were directly counted with a hemocytometer, and cell proliferation curves were plotted. Cell proliferation was also evaluated by EdU incorporation. Briefly, cells were seeded into 24-well plates in triplicate and treated with or without 10 μM cisplatin. After 72 h, cells were incubated with 10 μM EdU reagent (BeyoClick EdU-594, Beyotime, Haimen, China) for 2 h at 37°C. Nuclei were stained with Hoechst 33342 (Beyotime) for 30 min. The stained cells were photographed under an inverted fluorescent microscope. The percentage of EdU-positive cells was determined.

A total of 1.2 × 10⁶ CMECs were plated in confocal dishes. Fluorescent images of each cell sample were obtained via TCS SP8 laser confocal microscopy (Leica, Germany) and quantitatively analysed using ImageJ. BeyoClick™ EdU-594 (Beyotime, China) was used to evaluate CMEC proliferation. After incubation with EdU for 2 h, the cells were fixed, permeabilized and stained with EdU colour-substrate solution. A TUNEL Apoptosis Assay Kit (Roche, Germany) was used to evaluate TUNEL-positive CMECs. According to the manufacturer's instructions, CMECs were fixed, permeabilized, and then stained with TUNEL detection solution in the dark for 60 min. An ROS assay kit (Beyotime, China) and a mitochondrial superoxide indicator (Invitrogen, USA) were utilized to label intracellular ROS and mitochondrial ROS (mtROS). The cells were rinsed and incubated with the two fluorescent probes in the dark at 37 °C for 20 min. The intracellular ROS and mtROS contents are expressed as the area of intensity (AOI). Mitochondrial membrane potential (MMP) depolarization was assessed by a JC-1 fluorescent probe and is represented as the ratio of red to green fluorescence intensity. Mitochondria and nuclei were labelled with MitoTracker Red solution and DAPI solution.