Forced expression of miR-125b-5p or knockdown of ATXN1 gene increases migration, invasion capability, and cancer stemness of HCC cell lines. (a) Representative image of the wound-healing assay in PLC/PRF5-miNC, PLC/PRF5-miR125b, and PLC/PRF5-R2-miNC cells (upper panel). The average migration distance was calculated from the wound healing area, and the time-dependent distance of migration is depicted in the lower panel (mean ± SD; n = 3). (b) Similarly, representative images of the wound-healing assay and time-dependent distance of migration in PLC/PRF5-siNC, PLC/PRF5-siATXN1, and PLC/PRF5-R2-siNC cells are shown in the lower panel (mean ± SD; n = 3). (c) Representative images of the invasion assay in PLC/PRF5-miNC, PLC/PRF5-miR125b, and PLC/PRF5-R2-miNC cells (left panel). The number of invading cells was counted, and the average numbers of invading cells are shown (right panel). (d) Similarly, representative invasion assay images and quantified invading cells in PLC/PRF5-siNC, PLC/PRF5-siATXN1, and PLC/PRF5-R2-siNC cells are shown. (e) Expression of CD44 and CD133 in PLC/PRF5-miNC and PLC/PRF5-miR125b cells was evaluated by two-color flow cytometry using mouse anti-human CD44 monoclonal antibody (APC) and mouse anti-human CD133 monoclonal antibody (PE). The percentage of CD44 + CD133 + cells based on the analysis of 10,000 cells is indicated. (f) Expression of CD44 and CD133 in PLC/PRF5-siNC and PLC/PRF5-siATXN1 cells was analyzed by flow cytometry and the percentage of CD44 + CD133 + cells was determined. * p < 0.05.