Na1.4 inverted dmi6000 microscope

Fluorescence-conjugated EGFR-AS1 or EGFR mRNA probes were used for RNA FISH, which was performed as previously described^{21 (link)}. Hybridization was performed using DNA probe sets (Ribobio, Guangzhou, China) according to the manufacturer’s instructions, and control cells were observed using an NA1.4 inverted Leica DMI6000 microscope (Leica, Heidelberg, Germany). The images were recorded using a Hamamatsu ORCA-R2 camera (Hamamatsu Photonics, Hamamatsu, Japan) and recorded with LAS AF software (Leica). RNA FISH experiments were performed in two RCC cell lines.

In situ hybridization was performed with a FISH kit (RiboBio, Guangzhou, China). Cells, frozen HCC sections, and paired adjacent liver tissues were briefly rinsed in PBS and fixed in 4% formaldehyde for 10 min. The cells were then permeabilized in PBS containing 0.5% Triton X 100 at 4 °C for 5 min, washed with PBS three times for 5 min, and prehybridized at 37 °C for 30 min before hybridization. Next, an anti- hsa_circ_101555, anti-U6, or anti-18S oligodeoxynucleotide probe (RiboBio, Guangzhou, China) was used in the hybridization solution at 37 °C overnight in the dark. The next day, cells were counterstained with DAPI and imaged using a NA1.4 inverted Leica DMI6000 microscope (Leica, Heidelberg, Germany). Images were captured using a Hamamatsu ORCA-R2 camera (Hamamatsu, Japan) and recorded using LAS AF software (Leica). The experiments were conducted in triplicate.