Fig 1: Gankyrin/STAT3/CCL24/CCR3 forms a positive autocrine-regulatory loop in ccRCC.a Luciferase assays were used to determine the transcriptional activity of CCL24 in 786-O or 769-P cells with or without gankyrin knockdown. b The STAT3-binding sites in the CCL24 promoter in 786-O or 769-P cells were blocked using reporter constructs harboring mutant STAT3 variants, and luciferase assays were performed to determine the transcriptional activity of CCL24 in 786-O or 769-P cells with or without gankyrin overexpression in the presence of the wild-type or mutant STAT3 plasmid. c Gankyrin-interacting proteins were identified by nano-LC-ESI-MS/MS, and the STRING protein–protein interaction network is presented. d Western blot assays revealed that endogenous gankyrin coimmunoprecipitated (co-IP) with endogenous STAT3 in 786-O cells. IgG served as the control for co-IP. e An ELISA was performed to determine the concentration of CCL24 in the CM from 786-O or 769-P cells with or without gankyrin overexpression in the absence and presence of STAT3 knockdown. f A ChIP-PCR analysis was performed to determine the binding of STAT3 to the promoter of CCL24 in 786-O cells. g Real-time PCR was used to determine the expression of PSMD10 (gankyrin) mRNA in 786-O or 769-P cells treated with human recombinant CCL24 protein (5 ng/ml) for 3 and 5 days in the absence or presence of SB328437 (10 ng/ml). h Real-time PCR assays were performed to examine the expression of PSMD10 (gankyrin) mRNA in 786-O or 769-P cells treated with human recombinant CCL24 protein (3, 5 ng/ml) for 3 days in the absence or presence of SB328437 (10 ng/ml). i Western blot assays were used to detect the protein expression of gankyrin, p-STAT3, and STAT3 in 786-O cells treated with human recombinant CCL24 protein (5 ng/ml) for 3 and 5 days in the absence or presence of SB328437 (10 ng/ml). j Western blot assays were performed to detect the protein expression of gankyrin, p-STAT3, and STAT3 in 786-O cells treated with human recombinant CCL24 protein (3, 5 ng/ml) for 3 days in the absence or presence of SB328437 (10 ng/ml). k Immunoprecipitation assays were employed to examine the binding of gankyrin to STAT3 in 786-O cells treated with human recombinant CCL24 protein (5 ng/ml) for 3 days in the absence or presence of SB328437 (10 ng/ml). All the data are presented as the means ± SDs, *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 2: Blocking the positive autocrine-regulatory loop ameliorates pazopanib resistance and inhibits lung metastasis of ccRCC.a Images of the luciferase intensity and orthotopic xenografts from different groups are presented (n = 5/group). The photon flux levels in the different groups of mice were examined, and the results are presented as the fold increases in tumor growth. b Representative images of H&E and IHC staining for gankyrin, STAT3, CCL24, and Ki-67 in tumor specimens from mice in the four groups were shown (scale bar = 50 µm). c The results from the IHC staining for gankyrin, STAT3, and CCL24 were evaluated through the H-score, and the results from the IHC staining for Ki-67 were evaluated by the percentage of positive cells (scale bar = 50 µm). d Images of the luciferase intensity and lung metastases from the different groups are presented (n = 3/group). The photon flux levels in the different groups of mice were examined, and the results are presented as fold increases in tumor growth and lung metastases. e Representative images of H&E and IHC staining for gankyrin, STAT3, CCL24, and vimentin in lung metastases from mice in the four groups were presented (scale bar = 20 µm). f The IHC staining scores for gankyrin, STAT3, CCL24, and vimentin in lung metastases from mice in the different groups are presented (scale bar = 20 µm). All the data are presented as the means ± SDs, *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 3: CCL24 exerts a protumoral role in ccRCC, and high CCL24 expression in ccRCC patients predicts poor postoperative prognosis.a 786-O or 769-P cells were exposed to a range of concentrations of human recombinant CCL24 protein (1, 3, and 5 ng/ml) for 3 days, and the viability of the ccRCC cells was determined by CCK-8 assays. b 786-O or 769-P cells were treated with human recombinant CCL24 protein (3 or 5 ng/ml) in the absence or presence of SB328437 (10 ng/ml) for 3 days, and the viability of the cells was determined using CCK-8 assays. The data are presented as fold changes relative to the naive group. c The percentage of apoptotic 786-O or 769-P cells treated with human recombinant CCL24 protein (3 or 5 ng/ml) in the absence or presence of SB328437 (10 ng/ml) for 3 days was analyzed by flow cytometry assays. d, e Representative images and the statistical analysis of the results from the invasion (d) and migration (e) assays of 786-O or 769-P cells treated with human recombinant CCL24 protein (3 or 5 ng/mL) in the absence or presence of SB328437 (10 ng/ml) for 3 days (scale bar = 200 µm) are shown. f The CCL24 concentration in the blood serum of healthy individuals (n = 15) and patients with localized ccRCC (n = 50), and metastatic ccRCC (n = 20) was determined by ELISA. g Representative images of H&E and IHC staining for CCL24 of ccRCC tissues and matched adjacent tissues are presented (n = 256; scale bar = 50 µm). h Representative images of H&E and IHC for CCL24 staining and statistical charts in ccRCC patients with high (n = 46) and low (n = 210) TNM stages are presented (scale bar = 50 µm). i A time-dependent receiver-operating characteristic (ROC) analysis was performed to examine the optimal H-score cutoff value for CCL24 in the training cohort (n = 128). j Representative images of H&E and IHC staining for CCL24 in ccRCC specimens are shown (scale bar = 50 µm). k, l Kaplan–Meier analyses of the OS and PFS of ccRCC patients were performed with the training cohort (n = 128) (k), and the validation cohort (n = 128) (l) (p-value: log-rank test). All the data are presented as the means ± SDs, *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 4: 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.
Fig 5: The combination of gankyrin, STAT3 or CCL24, and established indicators yields superior prognostic accuracy in predicting the prognosis of ccRCC patients.a Representative images of H&E staining and IHC staining for gankyrin and STAT3 in ccRCC tissues are presented (scale bar = 50 µm), and the results from the correlation analysis between gankyrin and STAT3 expression in the ccRCC samples are shown. b, c A time-dependent ROC curve analysis with the training cohort was performed to examine the optimal H-score cutoff value for gankyrin or STAT3 (n = 128). d According to the H-scores for gankyrin and STAT3 in ccRCC specimens, the patients were divided into four groups. Kaplan–Meier analyses of the OS and PFS of ccRCC patients in the training cohort are shown. e According to the H-scores for gankyrin and CCL24 in ccRCC specimens, the patients were divided into four groups. Kaplan–Meier analyses of the OS and PFS of ccRCC patients in the training cohort are presented. f Schematic diagram of the underlying mechanisms described in our study and the clinical significance of our findings. All the data are presented as the means ± SDs, *P < 0.05, **P < 0.01, and ***P < 0.001.
Supplier Page from Abcam for Anti-Eotaxin 2 antibody