Fig. 4

Overexpression of ZBTB46 leads to higher percentage of cells in the G₀/G₁ phase, lower percentage of cells in the S phase, and lower number of Ki67-positive cells, but no significant change in apoptosis or senescence. HAECs were infected with adenovirus transducing ZBTB46 or GFP control. Transduced HAECs were seeded at simliar sub-confluent density 24 h after infection, alongside non-treated cells as baseline control and assayed after 2 days. a Representative flow cytometry histogram of cell populations assigned to different cell cycle phases (G₀/G₁, S, and G₂/M) based on intensity of PI staining (reflecting DNA content), gated on single cell population. b Flow cytometry quantification of cell numbers (% of total cells) in each cell cycle phase. *P < 0.05, compared to GFP control group, one-way ANOVA, n = 4–6/group. c Frequency of Annexin V-positive cells (early apoptotic cells) using flow cytometry (gated on live GFP+ cells) showed no significant difference among groups. d Representative images of adenovirus-infected HAECs stained with Ki67 (red, marker of proliferation) and DAPI (blue, nuclei). e Quantification of Ki67-positive cells as percentage of total GFP+ cells using ImageJ software. ***P < 0.001, two-tailed t-test, n = 6–8/group. f Representative images staining for SA-β-galactosidase in different groups of HAECs. HAECs were exposed to repeated 5% ethanol treatment to induce senescence as a positive control (Positive C) for the assay. g Quantification of SA-β-gal-positive cells (% of total cells) in different groups of cells, manually counted and averaged from five field of views in each well. ***P < 0.001, compared to GFP control, one-way ANOVA, n = 4