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: Metformin treatment leads to downregulation of 30S and 26S proteasome assembly. (a) Representative in-gel chymotrypsin-like substrate activity assay of native cell lysates from Jeko-1 and H1299 cells treated with 10 mM metformin for 72 h (left panels) followed by immunostaining of 20S alpha 1-7 subunits of blotted native gels (right panel). Quantification of 30S, 26S, and 20S is presented as mean + SEM relative to the untreated control. Significance was determined using Student’s one-sample t-test. (b) Representative Western blot analysis of 26S assembly factors (S5b, p28, and Rpn6) and 20S (alpha 1-7 subunit) as well as of phosphorylated and total p70 S6 kinase (S6k) expression in Jeko-1 and H1299 cells treated with 10 mM metformin for 72 h. Beta-actin was used as loading control. Bar graphs of densitometric analysis show mean + SEM relative to the untreated control. Significance was determined using Student’s one-sample t-test. * p < 0.05; ** p < 0.01.
Fig 3: miR-1248 regulated the PSMD10 expression to influence the proliferation, invasion, and migration of CRC cells. Cell proliferation, invasion, and migration of SW480 cells in the anti-miR-NC, anti-miR-1248, and anti-miR1248 + shPSMD10 were assessed by CCK-8 assay (A), colony formation assay (C), and Transwell assay (E, G). Cell proliferation, invasion, or migration of SW620 cells transfected with miR-NC, miR-1248, or miR1248 + PSMD10 were assessed using CCK-8 assay (B), colony formation assay (D), and Transwell assays (F, H). Mean ± standard deviation values from three independent experiments are presented. *P < 0.05
Fig 4: hsa-miR-1248 directly binds with PSMD10 and regulates its expression in CRC cells. A Results of Droplet digital polymerase chain reaction (ddPCR) assay showing the mRNA expression of PSMD10 in the Normal, M_low, and M_High tissues; each group contained three samples. B Sequences of Hsa-miR-1248, PSMD10-WT, and PSMD10-Mut; (C) Results of dual-luciferase assay showing the relative luciferase activity in SW480 cells transfected either with PSMD10-WT or PSMD10-Mut. D Results of dual-luciferase assay showing the relative luciferase activity in SW620 cells transfected either with PSMD10-WT or PSMD10-Mut; (E) Results of quantitative real-time polymerase chain reaction showing the expression of PSMD10 in SW480 cells transfected with anti-miR-1248 or anti-miR-NC. F Expression of PSMD10 in SW620 cells transfected with miR-1248 or miR-NC; (G) Western blots showing the expression of PSMD10; a-tubulin served as the loading control (The edges of the blots are cropping, and the full-length blots are presented in Supplementary Fig. 1). The results in (C-F) are presented as mean ± standard deviation of three independent experiments. *P < 0.05
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