Fig 2: SEPT6 promotes HCC cell migration and invasion. Transwell assays were conducted to assess (A) MHCC-97H and (B) Huh7 cell migration and invasion (magnification, ×100). MMP2 and MMP9 (C) mRNA and (D) protein expression levels were measured via reverse transcription-quantitative PCR and western blotting, respectively. ***P<0.001 vs. shcontrol or Vector. SEPT6, septin 6; HCC, hepatocellular carcinoma; MMP, matrix metallopeptidase; sh, short hairpin RNA.

Fig 3: SEPT6 is upregulated in human HCC and predicts poor prognosis. (A) SEPT6 mRNA expression levels in 64 paired HCC samples and adjacent non-tumor tissues were assessed via RT-qPCR. (B) SEPT6 protein expression levels in HCC samples (n=4) and corresponding adjacent non-tumor samples (n=4) were assessed via western blotting. (C) Association between SEPT6 expression and overall survival as determined by Kaplan-Meier survival analysis. SEPT6 low expression (n=18) and SEPT6 high expression (n=46). (D) SEPT6 staining in HCC samples and corresponding adjacent non-tumor samples (magnification, ×100). (E) SEPT6 expression in HCC and corresponding adjacent non-tumor samples derived from the GEPIA database. SEPT6 (F) mRNA and (G) protein expression levels in the normal hepatocyte cell lines and HCC cell lines were determined via RT-qPCR and western blotting, respectively. *P<0.05, **P<0.01 and ***P<0.001 vs. N or THLE2. SEPT6, septin 6; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative PCR; GEPIA, Gene Expression Profiling Interactive Analysis; N, adjacent non-tumor tissues; C, HCC tissues.

Fig 4: SEPT6 regulates the Hippo/YAP signaling pathway in HCC. (A) p-LATS1, LATS1, p-YAP and YAP protein expression levels were measured via western blotting. (B) Nuclear YAP protein expression levels were measured via western blotting using Lamin B1 as the loading control. (C) Co-immunoprecipitation assays were performed to determine the interaction between SEPT6 and LATS1 or LATS2. SEPT6-overexpression Huh7 cells were treated with 10 µM Lat. B for 2 h to disrupt the cytoskeleton. (D) Protein expression levels of p-LATS1, LATS1, p-YAP and YAP were measured via western blotting. (E) F-actin formation was determined by performing immunofluorescence assays (magnification, ×400). *P<0.05, **P<0.01 and ***P<0.001 vs. shcontrol or Vector; #P<0.05 and ##P<0.01 vs. SEPT6. SEPT6, septin 6; YAP, yes-associated protein; HCC, hepatocellular carcinoma; p, phosphorylated; LATS1, large tumor suppressor kinase 1; Lat. B, Latrunculin B; t, total; sh, short hairpin RNA.

Fig 5: SEPT6 promotes HCC cell proliferation. SEPT6-knockdown MHCC-97H and SEPT6-overexpression Huh7 cells were established by transfection with the corresponding plasmids. At 48 h post-transfection, cells were treated with G418 (400 µg/ml) for 2 weeks for stable cell selection. (A) Transfection efficiencies were determined via RT-qPCR. (B) Cell Counting Kit-8 assays were performed to assess cell proliferation. (C) Transfection efficiencies were also determined via western blotting. (D) Flow cytometry was conducted to analyze cell cycle distribution. Cyclin D1 and cyclin E1 (E) mRNA and (F) protein expression levels were measured via RT-qPCR and western blotting, respectively. *P<0.05, **P<0.01 and ***P<0.001 vs. shcontrol or Vector. SEPT6, septin 6; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin RNA.