Hoechst 33342

The assay of cell proliferation was performed with Cell-Light Apollo567 In Vitro Kit (RiboBio, Guangzhou, China). Complete medium was used to re-suspend the Schwann cells that were then tallied and plated on 96-well poly-L-lysine-coated plates. EdU was applied and the cells were cultured after cell transfection. The cells were fixed with phosphate buffered saline containing 4% formaldehyde and stained with Apollo 567 (RiboBio, Guangzhou, China) and Hoechst 33342 (RiboBio). Schwann cell proliferation analysis was performed using randomly selected images through a fluorescence microscope (Leica, Mannheim, Germany). The proliferating cell numbers were calculated. The average number of proliferating cells in the control group was set as 100%. The cell proliferation rate of p-GV230-Claudin-15 group was obtained by dividing by the average number of proliferating cells in the negative control or pGV230 group. The results were presented as fold change.

In total, 1×10⁵ SPC-A1 cells were cultured on coverslips in triplicate in 24-well plates and treated with 0, 2.5, 5 or 10 µM #2714 for 48 h as aforementioned. Cell proliferation was determined using an EdU incorporation assay kit, in which Hoechst 33342 was included (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's protocol. Briefly, following treatment with #2714, cells were incubated with 500 µl EdU solution (50 µM) diluted with completed medium at 37°C in darkness for 2 h, then fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 500 µl Hoechst 33342 solution at room temperature in darkness for a further 30 min. The percentage of proliferating cells was determined using an inverted fluorescence microscope (magnification, ×40; Zeiss AG, Oberkochen, Germany).

After the 8-week sampling period, 10 mg of EdU (RiboBio, Guangzhou, China) was dissolved with phosphate buffered saline (PBS) solution to make a 1 mg/mL stock solution. This dose is based on a study in mice (100 μg/g body weight; Guo et al., 2009 (link)). The DI of 10 fish from each group were harvested at 12 h after intraperitoneal injection and fixed with 4% paraformaldehyde for 24 h (Guo et al., 2009 (link)). Then, Apollo buffer (RiboBio) was used to incubate all fixed DI samples for 30 min away from light. These samples were washed with 0.5% TrixtonX-100 solution for 10 min, and then stained with Hoechst 33342 (RiboBio) for 30 min away from light. Images of these DI samples were captured using fluorescent microscopy (Nikon Eclipse Ci, Japan).

Cell Counting Kit-8 (CCK-8) and EdU assays were performed to measure cell proliferation. HASMCs were seeded into 96-well plates (2–3×10³ cells/well). Cells were incubated at 37°C in serum-free DMEM with or without 20 ng/ml PDGF-BB (R&D Systems, Inc., Minneapolis, MN, USA) for 24 h following transfection. For the CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assay, 10 µl CCK-8 solution was added to each well, and the absorbance was measured at 450 nm following 3 h of incubation at 37°C.
For the EdU (Guangzhou RiboBio Co., Ltd.) assay, the cells were incubated at 37°C in EdU solution (50 nmol/l) for 2 h, and fixed at room temperature in 4% formaldehyde for 30 min. The cells were subsequently exposed to 1,000 µl Cell-Light™ EdU Apollo^®488 In Vitro Imaging kit (100T) (938 µl dH₂O, 50 µl reaction buffer, 10 µl catalyst solution, 3 µl fluorescent dye solution, 9 mg buffer additive) for 30 min, followed by nuclear staining at room temperature with Hoechst 33,342 for 30 min (all Guangzhou RiboBio Co., Ltd.). An inverted fluorescence microscope was used to capture and analyze the images (magnification, ×100; Axio Observer Z1).

Schwann cells were seeded onto 96-well culture plates at a density of 2 × 10⁵ cells/mL and exposed to 50 μM 5-ethynyl-2′-deoxyuridine (EdU) using Cell-Light^TM EdU Apollo567 In Vitro Kit (Ribobio). At 24 hours after EdU exposure, Schwann cells were fixed with 4% paraformaldehyde and stained with Apollo dye solution and Hoechst 33342 (Ribobio). Images were obtained using a DMR fluorescence microscope (Leica Model DMi8, Leica Microsystems CMS GmbH, Bensheim, Germany). Number of proliferating cells and total cells were quantified using ImageJ (v1.8.0.112) (National Institutes of Health, Bethesda, MD, USA) (Schneider et al., 2012).