In our study, the animal test was authorized by the Animal Experimentation Ethics Committee of Jinan People’s Hospital. Six-week old BALB/c nude mice were obtained from the Shanghai Experimental Animal Center (SLAC, Shanghai, China). The mice in our study were freely divided into two groups with six in each group. A172 cells were transfected with sh-NC or sh-circEXOC6#1, which was subcutaneously injected into nude mice to structure the xenograft models. Once a week for 28 days, we measured the tumor volume. The tumor was weighed at 28 days of sacrificed mice. QRT-PCR or western blot assays were carried out to check the levels of circEXOC6, miR-433-3p, and FZD6.
In the immunohistochemistry (IHC) assay, tumors tissues were sectioned (5 μm thick) in paraffin. The primary antibody anti-FZD6 (Abcam, 1:1,000, ab150545) and the slices were co-incubated in a refrigerator overnight at 4°C after dewaxing and repair, and cultured with the secondary antibody (Abcam, 1:50,000, ab205718). After washing twice with the PBS solution, the slices were stained with diaminobenzidine for 3 min and subsequently re-dyed with hematoxylin (Beyotime). Finally, it was photographed under a light microscope (Nikon).
In the immunohistochemistry (IHC) assay, tumors tissues were sectioned (5 μm thick) in paraffin. The primary antibody anti-FZD6 (Abcam, 1:1,000, ab150545) and the slices were co-incubated in a refrigerator overnight at 4°C after dewaxing and repair, and cultured with the secondary antibody (Abcam, 1:50,000, ab205718). After washing twice with the PBS solution, the slices were stained with diaminobenzidine for 3 min and subsequently re-dyed with hematoxylin (Beyotime). Finally, it was photographed under a light microscope (Nikon).