Fig. 3: Gankyrin facilitates the growth and progression of ccRCC cells by promoting autocrine CCL24/CCR3.

a, b Cytokine profiles of conditioned medium (CM) from 786-O cells with/without gankyrin overexpression (a) or with/without gankyrin knockdown (b) and control 786-O cells were analyzed using a RayBiotech Human Cytokine Antibody Array. Heatmaps of the significantly differentially expressed cytokines are presented. c A Venn diagram of the significantly differentially expressed cytokines among the indicated groups is shown. d, e ELISAs were performed to determine the CCL24 concentration in the CM from the gankyrin-overexpressing and control 786-O or 769-P cells (d) and from the 786-O or 769-P cells with or without gankyrin knockdown (e). f Real-time PCR was performed to determine the expression of CCL24 mRNA in 786-O or 769-P cells with or without gankyrin overexpression in the absence and presence of STAT3 knockdown. g The mRNA expression of CCL24 in 786-O or 769-P cells with or without gankyrin knockdown was analyzed by real-time PCR. h CCK-8 assays were performed to determine the viability of 786-O or 769-P cells with or without gankyrin overexpression in the absence or presence of the CCL24 antibody (10 ng/ml) or SB328437 (10 ng/ml) at the indicated times. The data are presented as fold changes relative to the control group. i The percentage of apoptotic 786-O or 769-P cells with or without gankyrin overexpression in the absence or presence of the CCL24 antibody (10 ng/ml) or SB328437 (10 ng/ml) was analyzed by flow cytometry assays. j, k Representative images and statistical analysis of the results from the invasion (j) and migration (k) assays of 786-O and 769-P cells with or without gankyrin overexpression in the absence or presence of the CCL24 antibody (10 ng/ml) or SB328437 (10 ng/ml) are presented (scale bar = 200 µm). l, m 786-O cells with or without gankyrin overexpression in the absence or presence of the CCL24 antibody (10 ng/ml) or SB328437 (10 ng/ml) were treated with pazopanib (5 μM) for 36 h, and the resulting apoptosis was analyzed by flow cytometry assays. Cell viability was examined through CCK-8 assays. n, o A total of 5 × 10⁶ 786-O with or without gankyrin overexpression in the absence or presence of the CCL24 antibody (10 ng/ml) or SB328437 (10 ng/ml) were subcutaneously injected into nude mice (n = 5/group), and the tumor xenografts derived from the two groups are presented (n). The volumes of the tumor xenografts from the two groups were compared at the indicated times (o). All the data are presented as the means ± SDs, *P < 0.05, **P < 0.01, and ***P < 0.001.