Ribo fish kit

RNA FISH assay was performed with the Ribo FISH Kit (RiboBio, China) according to the manufacturer’s instructions. The specific procedure was performed in accordance with the manual provided with the kit. Cells were placed on confocal dishes and incubated at 37°C overnight. Cells were then washed and fixed with 4% paraformaldehyde. Paraformaldehyde-fixed cells were washed with PBS and blocked with blocking solution. Cells were hybridized with probes at 37°C overnight. The cells were then observed and photographed under a confocal fluorescence microscope. For the tumor tissue collected, RNA FISH probe labeling and RNA FISH procedures were performed as described previously [21 (link)].

For FISH, VSMCs were washed with 1× PBS solution three times at room temperature. The cells were fixed with 4% paraformaldehyde and 0.3% Triton X-100 in PBS mixture for 30 min and then washed with PBS. Pre-hybridization buffer was added at 37 °C for 30 min. Hybridization was carried out with a FISH probe in a moist chamber at 37 °C in the dark overnight using a Ribo FISH Kit (C10910, RiboBio, Guangzhou, China). The cells were washed with hybridization washing buffer at 42 °C. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA). For immunofluorescence, after two steps of fixing and permeabilization, the cells were blocked with 10% goat serum and then incubated with primary antibody at 4 °C overnight. The cells were washed with PBS and then incubated with Alexa Fluor 488 (1:500, anti-mouse, Cell Signaling Technology, Danvers, MA, USA) at room temperature for 2 h. The cellular location of NOVA1 was identified by immunofluorescence staining with NOVA1 antibody (1:100, novus). FISH was photographed by laser scanning confocal microscope (Olympus, FLUOVIEW FV1000) and the co-localization assay was photographed by Leica TCS SP8 STED.

Cells were inoculated in a 35-mm culture dish and maintained at 37 °C with 5% CO₂. LNCaP and Du 145 cells were rinsed with PBS and maintained for 15 min in 4% formaldehyde for fixing, they were then subjected to Triton X-100 and dehydrated with ethanol. The Cy3-labeled SNHG3 probe was bought from RiboBio (Guangzhou, China). Following the manufacturer’s guidelines, RNA FISH was conducted 3 times with a Ribo FISH kit (RiboBio RN: R11060.7) [34 (link)]. Finally, images were observed under a Zeiss LSM880 fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).