Hybridization buffer

Cells were cultured on coverslips, fixed and permeablized as previously described by us [14 (link)]. Subsequently, the coverslips were hybridized in hybridization buffer (Geneseed Biotech, Guangzhou, China) with digoxin (Dig) and biotin (Bio)-labeled single-stranded DNA probes at 37 °C overnight. Digoxin-labeled probes (Dig-5’-CATCTTTTCTGACACAGAGACGGCG-3′-Dig) specific to circPTK2 back-splice region and biotin-labeled probes against miR-429/miR-200b-3p (for miR-429, Bio-5’-ACGGTTTTACCAGACAGTATTA-3’-Bio; for miR-200b-3p, Bio-5’-TCATCATTACCAGGCAGTATTA-3’-Bio) were prepared (Geneseed Biotech). The signals were detected by Cy3-conjugated anti-digoxin and FITC-conjugated anti-biotin antibodies (Jackson ImmunoResearch Inc., West Grove, PA, USA). Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Finally, the images were obtained on a Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany). Each experiment was performed three times.

After the culturing on coverslips, fixation, and permeabilization, the coverslips went through hybridization overnight utilizing hybridization buffer (Geneseed Biotech, Guangzhou, China) with the addition of digoxin as well as biotin-labeled single-stranded DNA probes at 37°C. digoxin-labeled probes for circ-AKT1 were obtained from GenePharma (Shanghai, China). Detection of signals was conducted by using Cy3-conjugated anti-digoxin, as well as fluorescein isothiocyanate (FITC)-conjugated anti-biotin antibodies (Jackson ImmunoResearch, West Grove, PA, USA). Then cell nuclei underwent counterstaining by utilizing DAPI. Images were captured by utilizing Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany).

Cy3-labeled probe for circACTN4 was synthesized (Geneseed, Guangzhou, China). BC cells were cultured on coverslips, incubated with antibodies specific for FUBP1 (Thermo fisher scientific, USA) at 4 °C overnight, and treated with FITC-labeled goat anti-rabbit IgG (Bosterbio, Wuhan, China; 1:1000 dilution) at 37 °C for 2 h, and then incubated with FISH probe in hybridization buffer (Geneseed, Guangzhou, China) at 37 °C for 16 h. the cell nuclei were stained by DAPI(4′6- diamidino- 2- phenylindole). These images were taken with Olympus BX51 fluorescence microscope (Tokyo, Japan). The probe sequences were displayed in Additional file 1: Table S3