Bromodeoxyuridine (brdu)

A2780 cells (1×10⁴) were seeded into each well of a 6-well plate. At 24 h, the cells were treated with 10 µM BrdU (Beyotime Institute of Biotechnology) at 37°C for 1 h, fixed with 2% formaldehyde for 10 min at room temperature, washed with 1X PBS, permeabilized in 0.2% Triton X-100 for 10 min at room temperature, and washed with 1X PBS. The cells were incubated with a phycoerythrin-conjugated-BrdU antibody (cat. no. 50230; 1:500 dilution; Cell Signaling Technology, Inc.) at 37°C for 2 h. The cell nuclei were stained with DAPI (Beyotime Institute of Biotechnology). The positive cells were observed in four randomly selected fields under a BX51 fluorescence microscope.
Statistical analysis. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, Inc.) and data are presented as the mean ± standard deviation differences between two groups were evaluated using Student's t-test, and differences among multiple groups were evaluated using analysis of variance, followed by Tukey's multiple comparison test. The paired t-test was used for analyzing the IHC score in paired samples, and Wilcoxon signed-rank test was performed for TCGA data analysis. Kaplan-Meier followed by log-rank test was performed to analyze the rate of disease-free survival and overall survival. P<0.05 was considered to indicate a statistically significant difference.

Cell proliferation was evaluated by the BrdU incorporation assay. Briefly, SMMC7721 and HCC-LM3 cells transfected with different siRNAs were seeded on 6-well plates, and were incubated with 10 µM BrdU (Beyotime) for 4 hours followed by staining with mouse anti-BrdU antibody (Cell Signaling Technology) overnight in the dark at 4°C. After that, the cells were treated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Dako, Glostrup, Denmark). Propidium iodine (PI) (Sigma) (50 μg/mL) was used to stain nuclei as the control to all cells in each group. The labelling index was expressed as the number of positively labelled nuclei/total number of nuclei.

Cells were grown on coverslips. The cells were then stained with 10 µM BrdU (Beyotime Institute of Biotechnology) for 4 h at 37°C and were then incubated with BrdU mouse mAb (1:300; cat. no. 5292; Cell Signaling Technology, Inc.) for 1 h at room temperature. The cells were then stained with anti-mouse IgG Alexa Fluor 488 (1:300; cat. no. 4408, Cell Signaling Technology, Inc.) for 1 h at room temperature avoiding light. The nuclei were stained with DAPI for 5 min at room temperature. The cells were viewed using a confocal microscope (FV10i; Olympus Corporation).

Cells on coverslips were incubated with 10 μM BrdU (Beyotime) for 8 h at 37 °C, fixed with 4% paraformaldehyde for 30 min, and washed with PBS containing 1% Triton X-100 for 30 min. Then, the cells were incubated with 2 N HCl for 30 min at room temperature. After washing with PBS, the cells were blocked with PBS containing 1% Triton X-100 and 3% BSA. The cells were incubated with primary antibodies against BrdU (Abcam, ab6326, 1:50) overnight at 4 °C. Cells were further incubated with Alexa Fluor 647 (Jackson ImmunoResearch, 141562, 1:1000) for 1 h in the dark and then stained with DAPI (Beyotime). Each experiment was performed at least three times independently.

HUVECs were incubated with conditioned medium or conditioned medium containing EGCG (10, 30, 50 μm) or EGCG (50μm)+HMGB1 (5 ng/ml) for 24 h. Cell Counting Kit-8 (CCK-8; Beyotime, Shanghai, China) and BrdU (Beyotime, Shanghai, China) were used to determine cell proliferation according to the manufacturer’s instructions. For CCK-8 assay, HUVECs were incubated with 10 μl CCK-8 for 2 h, and absorbance at 450 nm was measured with a microplate reader (Bio-Rad, CA, USA). For BrdU cell proliferation assay, HUVECs were incubated with 5 μM BrdU for 4 h before 4% paraformaldehyde fixation. The BrdU signal was revealed by rat anti-BrdU antibody and goat anti-rat IgG. After DAPI staining, the proportion of BrdU-positive HUVECs was counted under the fluorescence microscope (Nikon, Japan).