Tcs sp2

Following the instructions provided with the EdU labelling/detection kit (RiboBio, Guangzhou, China), HepG2 cells were incubated in a final volume of 2 mL of complete medium at 2.5 × 10⁵ cells per well on a glass-bottom dish. Following incubation for 18 h, the medium above the cells was discarded and replaced with fresh medium containing PB at different concentrations (2.5 μM, 5 μM and 10 μM) or DMSO (<0.1%) as the vehicle. Twenty hours later, 100 μL of fresh medium containing of 50 μM EdU labelling agent was added to the cell cultures, which were then incubated for another 8 h at 37 °C under a 5% CO₂ atmosphere. Then, cells were incubated with glycine for 5 min after being fixed with 4% paraformaldehyde (pH 7.4) for 30 min. After being washed with PBS, the cells were stained with the anti-EdU working solution at room temperature for 30 min. Following a wash with 0.5% Triton X-100 in PBS, the cells were incubated with 5 μg/ml of Hoechst 33342 at room temperature for 30 min, and then, HepG2 cells were followed by observing them under a confocal laser scanning microscope (TCS SP2, Leica Microsystems, Germany; LSM710, Carl Zeiss, Germany).

To quantify cells of interest in immunostained sections, we obtained images of the stained sections by fluorescence microscopy (model TCS SP2; Leica Microsystems). Three slices per mouse and three fields from each slice were used for quantification; we manually outlined the cells and quantified them using the ImageJ software. The threshold values were maintained at a constant level for all analyses using the ImageJ software. ED1-positive, pSTAT3-positive, IBA1-positive, and NICD-positive areas were divided by the area of the image and are expressed as percentages. The number of NeuN-positive cells and activated caspase 3-positive cells was manually counted at × 400 magnification. The average number of activated caspase 3-positive cells expressing NeuN in these images was quantified.

This assay was performed with an Oris cell migration assay kit (Platypus Technologies, Fitchburg, WI, USA) according to the manufacturer’s instructions. Briefly, primary mouse lung fibroblasts were seeded (2.5 × 10⁴ cells/well) in an Oris Pro Cell Migration Assay 96-well tissue culture-treated plate. Two hours later, once the cells were adhered to the plates, the plate was incubated for an additional 12 h to allow cells to migrate. The wells were then washed with PBS and fixed with 4% paraformaldehyde (PF) for 15 min. After being washed 3 times, the wells were incubated with CoraLit® 594-phalloidin for 2 h at room temperature. Images were captured with a confocal microscope (TCS SP2, Leica Microsystems, Heidelberg, GmbH) utilizing a 4× objective.