Fig. 3

Inhibition of proliferation in HCC cells with reduced MDK expression. (A) Relative expression of MDK at the mRNA level in MHCC-97H cells after siRNA transfection. (B) Relative expression of MDK at the mRNA level in Huh7 cells after siRNA transfection. (C) Midkine protein levels in MHCC-97H cells after siRNA transfection. The grey values of the strips were quantified using ImageJ 2 software, defining the average grey value of siControl as 1. (D) Midkine protein levels in Huh7 cells after siRNA transfection. The grey values of the strips were quantified using ImageJ 2 software, defining the average grey value of siControl as 1. (E) Clone formation experiments were performed after the down-regulation of the MDK gene in MHCC-97H and Huh7 cells. The number of clonal clusters was quantified using ImageJ 2 software. (F) EdU staining experiments were conducted after the down-regulation of the MDK gene in MHCC-97H and Huh7 cell lines. Cells undergoing proliferation were marked with a green label, while nuclei were indicated with a blue label. The quantification of each fluorescent cell type was performed using ImageJ 2 software. (G) Following the knockdown of the MDK gene in MHCC-97H and Huh7 cells, the cells were subsequently cultured in 96-well plates. After a duration of 4 h, absorbances at 450 nm were measured using a microplate reader following CCK-8 staining. This measurement was recorded as the initial value on the first day. Absorbance readings were taken at 24-h intervals to assess cell viability. Compare the significance of the difference between each group and the siNC group using Student's t-test. Probability values p < .05 denote statistical differences, with statistical significance indicated by “*” for p < .05 and “**” for p < .01. Three independent replicate experiments were performed, four replicates per experiment for RT-qPCR and three replicate measurements per replicate for WB experiments. The quantification results are shown as the mean ± SD.