Cck 8 reagent

The phenotype assays of MGC803 cells were performed after transfected with eccDNA for 48 h. For CCK8 testing, approximately transfected 2 × 103 cells in 100 μl were incubated in quadruplicate in 96-well plates. At 0, 1, 2, 3 and 4 day, the CCK-8 reagent (Meilunbio) was added to each well and incubated at 37 °C for 2 h. Absorbance at 450 nm was recorded by the Synergy H1M Multimode microplate reader (BioTek).
For formation analysis, approximately transfected 1 × 103 cells in 3 ml were incubated in quadruplicate in 6-well plates. Cells were stained by crystal violet (Meilunbio) followed by incubating for two weeks. Image J software (version 1.8.0; National Institutes of Health) was used for calculating the cell cluster each well.
For EdU assay, approximately transfected 8 × 103 cells in 100 μl were incubated in quadruplicate in 96-well plates. Transfected cells were added with 50 mM EdU (Beyotime) and incubated for another 2 h following by incubation for 24 h at 37 °C and 5% CO₂. Cells were then fixed with 4% paraformaldehyde, permeated with 0.5 trition and stained with Apollo Dye Solution for proliferating cells. DAPI was used for labeling the Nucleic acids for all cells. Three randomly selected fields were taken using a fluorescence microscope. The cell proliferation rate was calculated using Image j software.

We seeded 1 × 10⁵ cells in six-well plates for fusion of 50–60% for 24 h; then cells were transfected with siRNAs mixed with siRNA-Mate (GenePharm) for 72 h and transfection efficiency was verified via RT-qPCR. We seeded 2000 transfected cells/well in 96-well plates, and then used CCK-8 reagent (MA0218-5, Meilunbio) to detect the viability of both cell lines at 24, 48, and 72 h. For plate colony formation assays, 500 transfected cells/well were added to six-well plates followed by treatment with CQ or phosphate-buffered saline (PBS) over the following 14 days. The colonies were finally fixed and stained with 0.1% crystal violet. All experiments were carried out with three triplicates. siRNAs targeting the back-splicing junction of hsa_circ_0001747 are shown in Supplemental Table 2.

Cell viability in vitro was evaluated by cell counting kit-8 (CCK-8) assay. 4 × 10³ cells were implanted into 96-well plates. After the cells adhered to the wall after 4 hours of culture, the medium in each well was replaced with 100 ml RPMI-1640 serum-free medium, and the adsorption rate after 2 hours was measured by a microplate reader at 450 nm in a medium containing 10 ml CCK-8 reagent (Meilunbio, MA0218-3). Then repeat the detection at 24 h, 48 h, and 72 h, respectively.

Transfected MIA PaCa-2 cells (1×10³ cells/well) and SW1990 cells (1.5×10³ cells/well) were seeded in 96-well plates. 100 μL DMEM containing 10 μL CCK-8 reagent (Meilunbio, Han Bio) was added to each well at 5 distinct time points. After incubation in the dark at 37°C for 2 h, absorption was detected at the 450 nm wavelength. Each group consisted of five duplicate wells, and the experiments were repeated in triplicate.