μ slide 8 well chamber slide

MA104 cell line (ATCC CRL-2378.1) or stably transfected MA104 cells (NSP5; Δ3, ΔT) were seeded into 24-well plates and subsequently infected with recombinant RVs at a multiplicity of infection (MOI) (FFU/cell) of 0.5 for multistep growth curve experiments and an MOI of 5 for a single-step growth curve experiment. After adsorption for 1 h at 37°C, the cells were washed twice with PBS and the medium was replaced with DMEM without trypsin. After incubation at 37°C, the cells were harvested after 8, 16, 24, and 36 h of virus adsorption. The cell lysates were freeze-thawed three times and activated with trypsin (1 μg/ml) for 30 min at 37°C. The lysates were used to infect monolayers of MA-NSP5 cells seeded in a μ-Slide 8-well chamber slide (iBidi GmbH, Munich, Germany). The cells were then fixed 5 h postinfection for 15 min with 4% paraformaldehyde and permeabilized for 5 min with PBS containing 0.01% Triton X-100. Next, cells were incubated for 30 min with PBS-BSA at room temperature and then with anti-NSP5 serum (1:1,000) diluted in PBS-BSA (22 (link)). After three washes with PBS, cells were incubated for 1 h at room temperature with TRITC-conjugated anti-guinea pig IgG (Jackson ImmunoResearch) (1:500) diluted in PBS containing 1% BSA (PBS-BSA).
The number of infected cells was counted, and the virus titers were expressed in focus-forming units per ml (FFU/ml).

Biotin-labeled probes or FAM-labeled probes were synthesized by Genepharma and used to visualize circARID1A or SLC7A5 in situ. Briefly, SGC7901 and BGC823 cells were seeded into μ-Slide 8-well chamber slide (Ibidi, Martinsried, Germany). The medium was removed 24 h later and cells were washed 2 × with PBS. Then cells were fixed with 4% paraformaldehyde for 10 min and treated with Triton X-100 (0.5%) at room temperature for 15 min. Cells were washed 2 × with PBS and incubated with denatured probes targeting circARID1A, SLC7A5, 18S rRNA or negative control and the slides were incubated at 37 °C overnight. The next day, nuclei were counterstained with DAPI and images were taken using a laser confocal microscope (Leica SP8, Wetzlar, Germany). The probe sequences for RNA-FISH are listed in Supporting Information Table S3.