Fig 2: miR-300 impacts on proliferation, apoptosis and cell cycle via regulating LEF-1 expression in hepatocellular carcinoma cells. (A) MTT assay was performed to detect the proliferation in Huh-7 cells co-transfected with LEF-1 vector (LEF-1) and miR-300 mimic (miR-300), as well as Hep3B cells co-transfected with LEF-1 shRNA and miR-300 inhibitor (n=3; *P<0.05, **P<0.01 vs. control). (B) The colony-forming ability was assessed using the colony formation assay. Representative images are shown. (C) The apoptosis rate was evaluated in Huh-7 and Hep3B cells by flow cytometric analysis following co-transfection with LEF-1 vector and miR-300/LEF-1 shRNA and miR-300 inhibitor (n=3; *P<0.05, **P<0.01). (D) The DNA content was analyzed using flow cytometry, and the percentage of cells in the G0/G1, S and G2/M phases of the cell cycle was calculated (n=3; *P<0.05, **P<0.01). OD, optical density; miR, microRNA; LEF-1, lymphoid enhancer-binding factor 1; shRNA, short hairpin RNA; 7-AAD, 7-aminoactinomycin D; PE, phycoerythrin.

Fig 3: LEF-1 enhances cell proliferation and inhibits apoptosis and cell cycle arrest in HCC cells. (A) Western blotting was used to detect the LEF-1 protein expression in the HCC cell lines Huh-7, Li-7, Hep3B and SNU-449, and the normal hepatic cell line L02. (B) RT-qPCR was used to detect the LEF-1 mRNA expression in the HCC cell lines Huh-7, Li-7, Hep3B and SUN-449, and the normal hepatic cell line L02 (n=3; **P<0.01, ***P<0.001 vs. L02). (C) The protein and mRNA expression of LEF-1 was evaluated in the Huh-7 cells transfected with LV-LEF-1 using western blotting and RT-qPCR analysis, respectively (n=3; ***P<0.001 vs. control). (D) The protein and mRNA expression of LEF-1 was detected in the Hep3B cells transfected with three LEF-1 shRNAs using western blotting and RT-qPCR analysis, respectively (n=3; ***P<0.001 vs. control). (E) MTT assay was performed to detect the proliferation of Huh-7 cells transfected with LV-LEF-1 and Hep3B cells transfected with LEF-1 shRNA (n=3; *P<0.05, **P<0.01 vs. control). (F) The colony-forming ability was assessed using the colony formation assay. Representative images are shown. (G) Flow cytometric analysis was used to observe the apoptosis rate of Huh-7 and Hep3B cells following transfection with LV-LEF-1 or LEF-1 shRNA, respectively (n=3; **P<0.01 vs. control). (H) The DNA content was analyzed in Huh-7 and Hep3B cells transfected with LV-LEF-1 or LEF-1 shRNA using flow cytometry, and the percentage of cells in the G0/G1, S and G2/M phases of the cell cycle was calculated (n=3; **P<0.01). HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; LEF-1, lymphoid enhancer-binding factor 1; LV, lentivirus; shRNA, short hairpin RNA: 7-AAD, 7-aminoactinomycin D; PE, phycoerythrin.

Fig 4: Association between LEF1 mRNA expression with survival status of MM patients. A, The overall survival curve exhibited the overall survival of MM patients with low or high expression levels of LEF1 mRNA using GenomicScape software (www.genomicscape.com). B, The progression-free survival (Left) and overall survival (Right) status of MM patients with low or high expression levels of LEF1 mRNA using GenomicScape software (www.genomicscape.com). Red line: high expression group, blue line: low expression group. P values were denoted on the plot.

Fig 5: Depletion of LEF1 repressed NF-κB signaling pathway through modulation of CYLD in U266 cells. A, The protein levels of p65 and p-p65 in U266 cells following LEF1 knockdown for 72 h was measured by Westernblot analysis. B, U266 cells transduced with shLEF1 or shCon were co-transfected with NF-κB reporter construct (p-NF-κB-luc) together with the pRL-CMV plasmid for normalization of transfection efficacy for 24 h, and relative luciferase activity was determined using dual-luciferase reporter assay kit. C, The mRNA expression levels of Cyclin D1, Bcl-2 and Bcl-xL in U266 cells transduced with shLEF1 or shCon for 72 h were determined using qRT-PCR. *P < 0.05 and **P < 0.01 versus shCon group. D, U266 cells transduced with shLEF1 or shCon were co-transfected with NF-κB reporter construct (p-NF-κB-luc) in the presence of CYLD siRNA or the control siRNA together with the pRL-CMV plasmid for normalization of transfection efficacy for 24 h, and relative luciferase activity was determined using dual-luciferase reporter assay kit. *P < 0.05 and **P < 0.01.