Cy5 streptavidin conjugate

The biotin‐labeled circ‐TNRC6B probe was purchased from Geneseed Biotech Corporation. The 5′ carboxyfluorescein (5′ FAM)‐modified miR‐452‐5p probe was designed and synthesized by GenePharma Corporation (Shanghai, China). The probe sequences are shown in Table S1. To perform cellular fluorescence in situ hybridization (FISH), KYSE150, and KYSE170 cells were seeded on coverslips and cultured for 12 h. The cells were rinsed with PBS and fixed with 4% paraformaldehyde. Next, the samples were treated with 0.2% Triton X‐100 on ice. The permeabilized samples were incubated with a blocking solution to block the nonspecific sites and hybridized with the probes for 12 h at 37 °C. The fluorescence signal of circ‐TNRC6B was detected with Cy5‐streptavidin conjugate (Invitrogen). The nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) for 10 min. To perform tissue FISH, ESCC chip array tissue slides were deparaffinized in xylene and ethanol solutions, followed by treatment with proteinase K at 37 °C for 10 min before blocking. The subsequent procedures were similar to those used in the cellular FISH analysis. The images were snapped using an LSM 900 confocal microscope (ZEISS, Oberkochen, Germany).