Fig. 6.

Upregulation of HHLA2 inhibits immune-independent ovarian cancer cells proliferation. a Western blotting of HHLA2 expression in ovarian cancer cell lines. The quantitative fold change in expression of HHLA2 normalized to GAPDH expression levels. The column on the right represented the relative expression level of HHLA2 in different ovarian cancer cell lines by Gray analysis. b Western blotting analysis of HHLA2 expression in PEO1 and OVCAR-3 cells and their HHLA2-overexpression counterparts. HHLA2-overexpression cells were transfected with lentivirus. GAPDH was detected as a loading control. c The expression level of HHLA2 in PEO1 and OVCAR-3 cells and their HHLA2-overexpressing counterparts were verified by qRT-PCR. ACTIN was detected as internal control. e, f Cell viability was evaluated by MTT assay at 1–5 days after cell seeded. The black curve represented the parental cell lines, the red curve represented the HHLA2-overexpression cell lines. It revealed that overexpression of HHLA2 significantly decreases the growth rate of PEO1 and OVCAR-3 cells(*p < 0.05, ** p < 0.01). g The EdU proliferation assay was performed in the HHLA2-overexpressing cell lines and their corresponding control groups. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. h, i Overexpression of HHLA2 decreased the mean proportion of proliferating ovarian cancer cells. The blue and the red column represented the proportion of proliferating cells in control and the HHLA2-overecpression group, respectively. h.* p = 0.027; i* p = 0.029. Three independent experiments were conducted for each assay