3 3 diaminobenzidine (dab)

Cells were lysed in RIPA buffer (cat. no. BB-3201; BestBio). BCA protein assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology) was used for the determination of total protein concentration. A total of 40 µg of protein lysate was separated by 9% SDS-PAGE and transferred onto a PVDF membrane (EMD Millipore). The membranes were blocked with 5% non-fat dry milk for 2 h at room temperature. The membranes were incubated with a primary antibody at 4°C overnight, followed by incubation with a secondary antibody (HRP-conjugated goat anti-Rabbit IgG; 1:2,000; cat. no. ZB2301; OriGene Technologies, Inc.) for 1 h at 37°C. The protein signals were detected by DAB (OriGene Technologies, Inc.). Primary antibodies included, anti-E cadherin antibody (1:300; cat. no. PB0583; Wuhan Boster Biological Technology, Ltd.), anti-N cadherin antibody (1:200; cat. no. BM-1573; Wuhan Boster Biological Technology, Ltd.), vimentin (1:100; cat. no. BM0135; Wuhan Boster Biological Technology, Ltd.) and β-actin (1:1,000, cat. no. TA-09; OriGene Technologies, Inc.). The intensity of bands were calculated using Tanon 1600 Gel Imaging system (Tanon Sciences and Technology Co., Ltd.).

The expression of SEPT6 in HCC samples and corresponding adjacent non-tumor tissues were analyzed by IHC, as previously described (19 (link)). Briefly, paraffin-embedded slides were de-paraffinized in xylene and rehydrated using an alcohol gradient. Antigen retrieval was performed by heating samples in 0.01 mol/l citrate buffer (pH 6.0) for 15 min in a microwave. Subsequently, the slides were immersed in 3% H₂O₂ at room temperature for 15 min to eliminate the endogenous peroxidase. After washing three times with PBS, the sections were blocked using 10% goat serum (Boster Biological Technology) at room temperature for 30 min. Subsequently, the sections were incubated with an anti-SEPT6 (cat. no. 12805-1-AP; 1:100; ProteinTech Group, Inc.) at 4°C overnight. After washing three times with PBS, the sections were incubated with a biotinylated secondary antibody (cat. no. SP-9000; OriGene Technologies, Inc.) at 37°C for 1 h. After washing three times with PBS, peroxidase activity was visualized using DAB (OriGene Technologies, Inc.) at room temperature for ~10 sec. Then, the sections were counterstained with hematoxylin (OriGene Technologies, Inc.) at room temperature for ~1 min. Stained samples were visualized using an IX71 light microscope (Olympus Corporation; magnification, ×100).

Immunohistochemistry was applied to evaluate CENP-A protein expression levels. The paraffin-embedded, formalin-fixed, 4μm thick tissue sections were all dewaxed with xylene and dehydrated with alcohol, and then rinsed in PBS followed by antigen retrieval via high-pressure steam treatment in a citrate buffer (pH 6.0) for 20 mins. After incubation, with the primary rabbit anti-CENP-A monoclonal antibody at 1:400 dilution (Abcam, Cambridge, UK) held at 4 °C overnight, we washed the sections before incubating with a second antibody (SP9000; goat anti-mouse IgG; OriGene Technologies, Inc., Beijing, China) and stained with DAB (OriGene Technologies, Inc., Beijing, China). The nuclei were stained with a Mayer hematoxylin.
Two experienced pathologists performed blind analysis on all the slides. The rating method for CENP-A expression was based on staining strength and the proportion of positively stained tumor cells. In cases where the two pathologists gave differing scores, the final decision was made following discussion by two observers.
Staining strength and intensity was measured as follows: 0, negative; 1, weakly positive (+); 2, moderately positive (+ +); and 3, strongly positive (+ + +). Scores of 0 and 1+ were considered as negative for CENP-A upregulation, while scores of 2+ and 3+ were considered positive for CENP-A upregulation.