Apollo staining solution

The effect of ²⁹³EMNVs and ^H19EMNVs on the proliferation of HMEC-1 was measured using an EdU Cell Proliferation Kit-488 with flow cytometry (EdU-488, RiboBio, Guangzhou, China) following the manufacturer’s protocol. In brief, HMEC-1, at an initial density of 2 × 10⁴ cells/well, were seeded into 48-well plates and cultured in normal (5.56 mM glucose according to the ingredient list provided by the manufacturer) or high glucose (HG, 25 mM glucose) medium containing ²⁹³EMNVs (50 μg/mL) or ^H19EMNVs (50 μg/mL) for 24 h as pretreatment. Then 150 μL of specific culture medium, described above, mixed with 0.15 μL of EdU was added into each well and incubated at 37 °C. After incubation for 2 h, cells were digested, centrifuged, washed with DPBS twice and fixed with 4% paraformaldehyde for 15 min. After neutralizing with 2 mg/mL glycine, cells were washed with DPBS twice, permeabilized with 0.5% Triton X-100 for 10 min and washed with DPBS. Next, cells were resuspended using Apollo staining solution (RiboBio), and incubated for 10 min. Cells were washed three times using 0.5% TritonX-100 and resuspended with DPBS. Finally, cells were analyzed using an easyCyte™ flow cytometer (Merck-Millipore, Darmstadt, Germany).

The proliferative ability of SGC7901 cells after different transfection were determined by the EdU proliferation assay (RiboBio Inc.). Twenty-four hours after transfection, cells were incubated in 50 M EdU for 5 h, and fixed within 4% paraformaldehyde for 30 min at room temperature (RT). Then the cells were washed in PBS twice and permeabilized using PBS containing 0.5% Triton X-100 for 10 min. After extensive washes in PBS, the cells were incubated lucifugally in Apollo staining solution (RiboBio Inc.) for 30 min, then repeated permeation and wash, and incubated in Hoechst 33342 (1:100; RiboBio Inc.) for another 30 min at RT. All of the staining were performed in triplicate.

Cell proliferation assays were performed using the Cell-Light^TM EdU Apollo In Vitro Kit (RiboBio, Guangzhou, China). Cancer cells (1 × 10⁵) were seeded in 96-well plates and treated according to the experimental requirements. Subsequently, EdU medium was added and the cells were incubated for 2 h. After washing the cells twice with PBS, they were fixed with 4% PFA solution (Biosharp) for 30 min. Cells were then incubated with glycine (2 mg/ml; Biofroxx, Germany) for 5 min and permeabilized with 0.5% Triton X-100 (Beyotime) for 10 min. After adding 1 × Apollo^® staining solution (RiboBio) and incubating for 30 min at light avoidance and RT, the cells were washed three times with 0.5% TritonX-100. Finally, a 1 × Hoechst 33342 reaction solution (RiboBio) was added and incubated for 30 min with light avoidance at RT. The cells were imaged using a microscope (Leica Microsystems) and counted in four randomly selected fields.