A00 1 1102

According to the manufacturer instructions, the glucose uptake of HTR-8/SVneo cells was measured by a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose (2-NBDG) (Invitrogen, N13195). Briefly, the cells were cultured in a medium with a 100 μM 2-NBDG for 1 hour at 37°C. The cells were washed twice with PBS, digested with trypsin, and centrifuged for 5 min at 1500 rpm at 4°C. Then, the cells were suspended in 200 μl of PBS in each tube and immediately analyzed and quantified by flow cytometry (A00-1-1102, Beckman, USA).

To conduct cell cycle detection, the two cells were treated with 1.2 mL of pre-cooled 100% ethanol, resulting in a final concentration of 75%. Subsequently, the cells were fixed by overnight incubation at 4℃. After the cells were washed with PBS, they were centrifuged. Then, they were mixed with 150 µL of propidium iodide (PI, MB2920, Meilune, China) and stained for 30 min at 4℃. Different stages of the cell cycle have different DNA content, and PI can label DNA to determine which cycle the cell is in. Detection of stained cells with a flow cytometer (A00-1-1102, Beckman, USA), PI was excited by a 488 nm argon laser and detected through a 630 nm bandpass filter, collecting around 15,000 cells in FSC/SSC dot plots, and analyzing the percentage of cells in each cell cycle phase on the PI fluorescence histogram to determine positive cell staining. For apoptosis detection, SK-Hep-1 and HCC-LM3 cells were treated with trypsin (AWC0232, Abiowell, China). Following digestion, after washing the cells with PBS, approximately 3.2 × 10⁵ cells were collected for further analysis. Following the instruction provided by the apoptosis detection kit (KGA1030, KeyGEN BioTECH, Nanjing, China), the cell suspension was treated with Annexin V-FITC (5 µL) and PI (5 µL) successively. The mixture was then incubated at room temperature for 10 min in the absence of light before further analysis.

Samples were taken, each about 1 × 10⁶ cells were placed in a 1.5 mL centrifuge tube, the cells were resuspended with 200 μL PBS volume, α-SMA (PA5-77846, eBioscience) and s-100 (MA5-32625, eBioscience) antibodies (5 μL for all three antibodies) were added respectively. The aforementioned cells were washed with 1 mL PBS, incubated in the dark for 30 min, and repeated twice. α-SMA and s-100 expressions were detected by flow cytometry (A00-1-1102, Beckman) after the cells were resuspended by adding 200 μL PBS, respectively.

The expression level of Fcnb and the number of neutrophils and macrophages were detected by flow cytometry [40 ]. Lung tissue samples were digested to obtain cell suspension. Blood, BALF, and cell suspension were centrifugally stratified to obtain neutrophils and macrophages for re-suspension. Antibodies of LY-6G (11-9668-82, eBioscience, USA) and F4/80 (11-4801-82, eBioscience, USA) were added respectively to the suspensions of neutrophils and macrophages and incubated for 30 min away from light. Then, the mixture of Fcn B antibody (Ab70814, Abcam, UK) and Alexa Fluor 594-goat anti-rabbit IgG (A-11012, Invitrogen, USA) was added to the above cell suspensions and incubated for another 30 min away from light. After washing and suspension, the cells were detected by flow cytometry (A00-1-1102, Beckman, USA). In addition, ROS levels in lung tissues and MLE-12 were detected by flow cytometry. First, the tissue and cells were digested to obtain cell suspensions. The cell suspension was incubated with BODIPY™ 581/591 C11 fluorescent probe (A-11012, Invitrogen, USA) at 37 °C for 30 min and finally detected by flow cytometry.

Apoptosis was detected according to the instructions of the apoptosis kit (KGA1030, KeyGEN BioTECH). Briefly, cells were digested with EDTA-free trypsin and washed with PBS to a concentration of 3.2 × 10⁵ cells. Cells were suspended by adding 500 µL of Binding buffer. Subsequently, the percentage of apoptotic cells was assessed by flow cytometry (A00-1-1102, Beckman).