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Intracellular ROS levels were monitored using 2′,7′-dichlorofluorescein diacetate (DCFH-DA; cat. no. 170V; Beijing Vigorous Biotechnology Co., Ltd, China) to identify the role of ROS in Scu and Scu-treated exosomes. RBMVECs were seeded in 24 well plates at a density of 2×10⁵ cells/well and incubated overnight at 37°C. Cells were subsequently incubated with or without intervention drugs (Scu, CE and SE) for 48 h. The cells were then treated with DCFH-DA (5 µM) at 37°C for 90 min. Fluorescence microscopy (magnification, ×400; U-SPT; Olympus Corporation, Tokyo, Japan) was used to measure the fluorescence intensity of the treated cells.
RBMVECs (1×10⁴ in 200 µl) were seeded in quadruplicate in a 96-well plate and incubated with 2.5 mmol/l Hcy for 48 h in the presence or absence of intervention drugs (Scu, CE and SE). The cultured cells were washed three times with PBS (pH 7.4) and incubated in DMEM containing DCFH-DA (5 µM) at 37°C for 90 min. The cells were washed with prewarmed PBS and covered with 100 µl of DMEM. The fluorescence intensity (FI) of each well was measured using a microplate reader at 485/530 nm.

Cells were incubated with 2.5 mmol/l Hcy for 48 h in the presence or absence of intervention drugs (Scu, CE and SE). Briefly, cells were grown on cover slips, and 4% paraformaldehyde was used to fix the cells for 30 min at room temperature. Cells were subsequently blocked in 10% goat serum (Gibco; Thermo Fisher Scientific, Inc.) for 20 min at 37°C. Cells were treated with antibodies against CD63 (cat. no. o67346b; OmnimAbs, Alhambra, CA, USA), claudin-5 (cat. no. 6679g62), ZO1 (cat. no. 3268f94; both Affinity Biosciences, USA) and occludin (cat. no. o29813c; OmnimAbs) for 1 h at 37°C (1:50 dilution). Cells were subsequently incubated for 1 h at 37°C with corresponding secondary antibodies conjugated to FITC. Cells were subsequently washed three times with PBS and stained with DAPI (1 µg/ml in PBS) at 37°C in the dark. The coverslips were mounted and cells were observed under a light microscope (U-SPT; Olympus Corp., Tokyo, Japan). Data were analyzed by using Medical Image Analysis Software 16.0 (MIAS, Warrendale, WA, USA).

After fixing in 4% formaldehyde overnight, blocks of lung tissue were sectioned, deparaffinized, and rehydrated in graded xylene. This was followed by staining with the primary anti-KLF5 monoclonal antibody (1:2000, Abcam, Cambridge, UK). After washing with phosphate-buffered saline (PBS), the sections were incubated with secondary antibody (1:200) for 1 hour at room temperature. Pictures were taken under a microscope (Olympus U-SPT; Olympus Corp., Tokyo, Japan) with high resolution. After staining of tissue slices, pathological images were analysed by Image-Pro 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) to achieve the purpose of semi-quantitative analysis of intensity. Images were captured by the Olympus U-SPT system. Positive tissues were randomly collected from each slice, and the intensity was measured in grey values.