Ab191208

Cells grown on glass coverslips or tissue sections were fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were washed twice with PBS. Blocking buffer (DakoCytomation, Glostrup, Denmark) was added for 30 min, and samples were then stained with primary antibodies and goat anti-mouse IgG/Alexa Fluor (Bioss Antibodies, bs-0296G-AF647, bs-0368G-AF488, 1:200 dilution). The following primary antibodies were used: αSMA (Proteintech, 55135-1-AP, 1:200 dilution), CD34 (Abcam, ab81289, 1:200 dilution), KLF2 (Abcam, ab203591, 1:100 dilution), KLF4 (Abcam, ab106629, 1:100 dilution), and Claudin5 (Abcam, ab15106, 1:100 dilution), ZO-1 (Cell Signaling, #8193, 1:1000 dilution), occludin (Abcam, ab216327, 1:100 dilution), CK (Abcam, ab191208, 1:200 dilution).

Oligonucleotide-modified probe sequence for circSPIRE1, GALNT3 mRNA, and QKI mRNA was synthesized from GenePharma (China). Paraffin-embedded tissue blocks were cut into 2.5-μm sections and transferred to glass slides. FISH was performed in tissue sections using a fluorescence in situ hybridization (FISH) kit purchased from GenePharma (China) following the manufacturer’s protocol. The images were obtained using the OLYMPUS FV1000 confocal microscope (Japan). Fluorescence intensity was analyzed by ImageJ. Pearson’s correlation coefficient was analyzed by OLYMPUS FV1000 software. The probe sequences are shown in Supplementary data 4. For immunohistochemistry (IHC), deparaffinization, rehydration, antigen retrieval, and staining were conducted in order. Normal goat serum blocking buffer (no. ZLI-9056), Rabbit two-step kit (no. PV-6001), and DAB kit (no. ZLI-9017) were purchased from ZSGB-BIO (China). The following primary antibodies were used: CD34 (1:200 dilution, ab81289, Abcam), CK (1:200 dilution, ab191208, Abcam), ELAVL1 (1:250 dilution, ab200342, Abcam), GALNT3 (1:100 dilution, AP51768PU-N, Origene), QKI (1:200 dilution, A300-183A, BETHYL), E-cadherin (1:250 dilution, ab40772, Abcam), and anti-Giantin (1:100 dilution, ab37266, Abcam).