Osteopontin opn

The extent of muscularization of small arteries was assessed following immunochemistry staining for α‐smooth muscle actin (α‐SMA) (Abcam, Cambridge, UK). Meanwhile, smooth muscle cells of small lung arteries exhibiting proliferation were assessed by immunostaining with proliferating cell nuclear antigen (PCNA) (ProteinTech, Wuhan, China) and osteopontin (OPN) (ProteinTech, Wuhan, China). Furthermore, the expression of KLF4 and P‐AKT was assessed by immunostaining with antibodies against KLF4 (ProteinTech, Wuhan, China) and P‐AKT (Gene Tex, San Antonio, USA).

After treatment without or with CBD (2.5 μM) and TNF-α (50 ng/mL) for 3 days, DPSCs were fixed with 4% paraformaldehyde solution at 37 °C for 20 min, treated with QuickBlock™ Blocking Buffer (Beyotime, China) for 10 min. Expression of osteogenic/odontogenic markers was analyzed by immunofluorescence staining using primary antibodies Osteopontin (OPN, 1:100, Proteintech, Wuhan, China) and primary antibodies COL-I (1:100, Proteintech, Wuhan, China) at 4 °C overnight, followed by incubation with fluorophore-conjugated secondary antibodies (1:400, Proteintech, Wuhan, China) for 1 h at room temperature. DAPI staining for 10 min was carried out after secondary antibody incubation. The cytoskeleton was stained with Phalloidine. Staining was detected using fluorescent microscopy (Olympus, Tokyo, Japan). Image J software was used for quantification.