Journal: Theranostics

Article Title: FBXW7/GSK3β-mediated proline-rich 11 degradation promotes oxidative DNA damage and inhibits tumor progression in renal cell carcinoma

doi: 10.7150/thno.106018

Figure Lengend Snippet: GSK3β interacts with PRR11 to regulate PRR11 stability. (A) HA-PRR11 plasmid was overexpressed in 293T cells, and the results were analyzed by co-IP with HA antibody, followed by silver staining (top) and mass spectrometry (bottom). (B) Western blot analysis of PRR11 in ACHN cells after IP analysis with GSK3β antibody. (C) Western blot analysis of His-GSK3β, GST-PRR11 after GST pull-down assays. (D) Diagram of the truncation of PRR11 (top). Full-length or truncated mutants of HA-PRR11 and Flag-GSK3β plasmid were transfected into 293T cells, and Western blot analysis of Flag-GSK3β was performed after IP analysis with HA antibody (bottom). PR: Proline-rich domain; P: CDC4 Phosphodegron (CPD) motif; D-box: D-box motif; KEN box: KEN box motif. (E) MG132 (10 μM), λ-PPase (8 U/μL), or GSK3β inhibitors CHIR-99021 (10 μM) and LiCl (20 mM) were added to 293T cells transfected with HA-PRR111 and Flag-GSK3β, after which Western blot analysis of HA-PRR11 was performed. (F) After exposing 293T cells transfected with HA-PRR11, Flag-GSK3β-WT, or Flag-GSK3β-Mut (GSK3β-S9D and GSK3β-Y216A) to CHX (50 μg/mL) for the indicated durations, Western blot analysis was conducted for HA-PRR11 (left). Quantification of PRR11 half-life (right, n = 3 biologically independent experiments). (G) 293T cells transfected with HA-PRR11, Myc-Ub, Vector, Flag-GSK3β-WT, or Flag-GSK3β-Mut were incubated with MG132 (10 μM) for 6 h, and Western blot analysis of Myc-Ub was performed after IP analysis with HA antibody. (H) GSK3β phosphorylation recognition sequence was compared with the conserved PRR11 sequence (left). Design the corresponding PRR11 dephosphorylation mimic mutants (PRR11-1A: T287A/S291A, PRR11-2A: T326A/T330A, PRR11-1A/2A: T287A/S291A/T326A/T330A), PRR11 phosphorylation mimic mutants (PRR11-1D: T287D/S291D, PRR11-2D: T326D/T330D, PRR11-1D/2D: T287D/S291D/T326D/T330D) and PRR11 phosphorylation motif deletion mutants (PRR11-1Δ: I286_S291del, PRR11-2Δ: L325_T330del, PRR11-1Δ/2Δ: I286_S291del/L325_T330del) according to the conserved sequence recognized by PRR11 (right). (I) 293T cells were transfected with the indicated plasmids and Western blot analysis of HA-PRR11 was performed. (J) HA-PRR11-WT was phosphorylated in vitro with active GSK3β and ATP-γ-S, and immunoblotted after alkylation with PNBM. (K) HA-PRR11-WT and HA-PRR11-1A/2A were phosphorylated in vitro with active GSK3β and ATP-γ-S, and immunoblotted after alkylation with PNBM. (L) 293T cells transfected with Myc-Ub, HA-PRR11-WT, or HA-PRR11-1D/2D were treated with DMSO or CHIR-990211 (10 μM) and incubated with MG132 (10 μM) for 6 h before cell collection, and then Western blot analysis of Myc-Ub was performed after IP analysis with HA antibody. (M) 293T cells transfected with Myc-Ub, Flag-GSK3β, HA-PRR11-WT, or HA-PRR11-1A/2A were incubated with MG132 (10 μM) for 6 h, and Western blot analysis of Myc-Ub was performed after IP analysis with HA antibody. Protein levels were quantitatively detected with ImageJ software, and linear regression was used to analyze the protein half-life (F). Data are presented as mean values ± SD.

Article Snippet: The immunoprecipitates were incubated with human active GSK3β (G09-10H, SignalChem) for 1 h at 30°C in kinase assay buffer (25 mM Tris-HCl, 25 mM KCl, 5 mM MgCl 2 , 1 mM DTT) containing 500 μM ATP-γ-S (ab138911, Abcam).

Techniques: Plasmid Preparation, Co-Immunoprecipitation Assay, Silver Staining, Mass Spectrometry, Western Blot, Transfection, Incubation, Sequencing, De-Phosphorylation Assay, In Vitro, Software

Journal: Theranostics

Article Title: FBXW7/GSK3β-mediated proline-rich 11 degradation promotes oxidative DNA damage and inhibits tumor progression in renal cell carcinoma

doi: 10.7150/thno.106018

Figure Lengend Snippet: FBXW7 promotes ubiquitination and degradation of PRR11 via GSK3β-mediated phosphorylation. (A) HA-PRR11, Flag-FBXW7, or Flag-FBXW7+ siGSK3β were transfected into 293T cells. After IP analysis with Flag antibody, Western blot analysis was performed on HA-PRR11. (B) 293T cells transfected with siFBXW7-1 , Flag-GSK3β, or siFBXW7-1+ Flag-GSK3β were incubated with CHX (50 μg/mL) for indicated times, and then Western blot analysis was performed for PRR11 (left). Quantification of PRR11 half-life (right, n = 3 biologically independent experiments). (C) 293T cells transfected with HA-PRR11, Myc-Ub, Flag-FBXW7, or Flag-FBXW7+ siGSK3β were treated with MG132 (10 μM) for 6 h, and Western blot analysis of Myc-Ub was performed after IP analysis with HA antibody. (D) 293T cells were transfected with Myc-FBXW7, Flag-GSK3β, HA-PRR11-WT, or PRR11 dephosphorylation mimic mutants (HA-PRR11-1A, HA-PRR11-2A, and HA-PRR11-1A/2A), and Western blot analysis of PRR11 was performed. (E) 293T cells transfected with Myc-Ub, Flag-FBXW7, Flag-GSK3β, HA-PRR11-WT, or PRR11 dephosphorylation mimic mutants were treated with MG132 (10 μM) for 6 h, and Western blot analysis of Myc-Ub was performed after IP analysis with HA antibody. (F) Schematic illustration of FBXW7-GSK3β can only promote phosphorylated PRR11 degradation. Protein levels were quantitatively detected with ImageJ software, and linear regression was used to analyze the protein half-life (B).

Article Snippet: The immunoprecipitates were incubated with human active GSK3β (G09-10H, SignalChem) for 1 h at 30°C in kinase assay buffer (25 mM Tris-HCl, 25 mM KCl, 5 mM MgCl 2 , 1 mM DTT) containing 500 μM ATP-γ-S (ab138911, Abcam).

Techniques: Transfection, Western Blot, Incubation, De-Phosphorylation Assay, Software

Journal: Theranostics

Article Title: FBXW7/GSK3β-mediated proline-rich 11 degradation promotes oxidative DNA damage and inhibits tumor progression in renal cell carcinoma

doi: 10.7150/thno.106018

Figure Lengend Snippet: FBXW7/GSK3β-PRR11 axis activates the AKT pathway and AKT activation inhibits PRR11 degradation. (A) KEGG pathway enrichment analysis was performed on DEGs in the RNA-seq expression matrix of PRR11 silencing ACHN cells. KEGG analysis was performed using the R package "clusterProfiler". The p -value was calculated by two-tailed Fisher's exact test, and p < 0.05 was used as a screening criterion. (B) Protein levels of each important factor of the AKT signaling pathway were analyzed by Western blot analysis after PRR11 knockdown in RCC cells. (C) Western blot analysis of AKT phosphorylation levels after transfection of ACHN and Caki-1 cells with siGSK3β or/and siPRR11 . (D) AKT activity was measured in ACHN cells transfected with siFBXW7 (top) /siGSK3β (bottom) or/and siPRR11 (n = 3 biologically independent experiments). (E) Changes in PRR11 protein levels were analyzed by Western blot analysis after transfection of AKT-CA or AKT-DN in RCC cells. (F) AKT-CA or AKT-DN plasmid was transfected into ACHN cells. After IP analysis with PRR11 antibody, Western blot analysis was performed on GSK3β and FBXW7. (G) Western blot analysis of PRR11 after transfection of ACHN cells with Flag-GSK3β (top)/Flag-FBXW7 (bottom) or/and HA-AKT-CA. (H) 293T cells transfected with the Flag-GSK3β or/and HA-AKT-CA were treated with CHX (50 μg/mL) for the indicated durations, after which Western blot analysis was performed for PRR11 (left). Quantification of the PRR11 half-life (right, n = 3 biologically independent experiments). (I) 293T cells transfected with the Flag-FBXW7 or/and HA-AKT-CA were treated with CHX (50 μg/mL) for the indicated durations, after which Western blot analysis was performed for PRR11 (left). Quantification of the PRR11 half-life (right, n = 3 biologically independent experiments). (J) 293T cells transfected with HA-PRR11, Myc-Ub, Flag-GSK3β, or/and GFP-AKT-CA were incubated with MG132 (10 μM) for 6 h, and Western blot analysis of Myc-Ub was performed after IP analysis with HA antibody. (K) 293T cells transfected with HA-PRR11, Myc-Ub, Flag-FBXW7, or/and GFP-AKT-CA were incubated with MG132 (10 μM) for 6 h, and Western blot analysis of Myc-Ub was performed after IP analysis with HA antibody. Protein levels were quantitatively detected with ImageJ software, and linear regression was used to analyze the protein half-life (H-I). The p -values were calculated with one-way ANOVA with Tukey's multiple comparisons test (D). Data are presented as mean values ± SD.

Article Snippet: The immunoprecipitates were incubated with human active GSK3β (G09-10H, SignalChem) for 1 h at 30°C in kinase assay buffer (25 mM Tris-HCl, 25 mM KCl, 5 mM MgCl 2 , 1 mM DTT) containing 500 μM ATP-γ-S (ab138911, Abcam).

Techniques: Activation Assay, RNA Sequencing, Expressing, Two Tailed Test, Western Blot, Knockdown, Transfection, Activity Assay, Plasmid Preparation, Incubation, Software

Journal: Theranostics

Article Title: FBXW7/GSK3β-mediated proline-rich 11 degradation promotes oxidative DNA damage and inhibits tumor progression in renal cell carcinoma

doi: 10.7150/thno.106018

Figure Lengend Snippet: PRR11-AKT axis promotes RCC proliferation and metastasis in vivo . (A) A schematic diagram on the construction of in vivo models of tumor proliferation and metastasis and drug therapy. (B) Gross diagram of xenograft model from mice injected ACHN cells transfected with shNC or shPRR11 with or without SC79 treatment. (C) Tumor volume statistic for mice injected subcutaneously with ACHN cells transfected with shNC or shPRR11 and treated with or without SC79 (n = 8 per group). (D) 18 F-FDG PET/CT scans were used to assess tumor growth and malignancy in each group of mice. Each group of images is represented from left to right as follows: CT image, PET image, PET/CT fusion image. (E) Representative images of Ki-67 and γ-H2AX IHC staining of tumor tissue from each group of mice in the xenograft model (top), and their quantitative statistical plots (bottom, n = 4 per group). (F) GFP fluorescence intensity between groups (control vs. shPRR11 vs. SC79 vs. shPRR11 +SC79) in the model of tail vein injection lung metastasis established using ACHN cells (left). Average radiation efficiency in lung is evaluated by ImageJ software, and statistics are performed (right, n = 3 per group). (G) Representative images of H&E staining of mice lung tissues from each group. (H) Gross diagram of a popliteal lymph node metastasis model of mice injected with ACHN cells transfected with shNC or shPRR11 with or without SC79 treatment (left). Popliteal lymph node volume statistics for mice injected with ACHN cells transfected with shNC or shPRR11 and treated with or without SC79 (right, n = 5 per group). (I) MRI T2 axial scanning imaging of popliteal lymph nodes in mice. (J) Mechanistic diagram of this study. FBXW7-GSK3β mediates PRR11 degradation, thereby regulating the AKT pathway. In turn, AKT-GSK3β is involved in regulating PRR11 degradation, forming a positive feedback loop that regulates oxidative DNA damage and accelerates RCC progression. The p -values were calculated with one-way ANOVA with Tukey's multiple comparisons test (C, E-F, H). Data are presented as mean values ± SD.

Article Snippet: The immunoprecipitates were incubated with human active GSK3β (G09-10H, SignalChem) for 1 h at 30°C in kinase assay buffer (25 mM Tris-HCl, 25 mM KCl, 5 mM MgCl 2 , 1 mM DTT) containing 500 μM ATP-γ-S (ab138911, Abcam).

Techniques: In Vivo, Injection, Transfection, Positron Emission Tomography-Computed Tomography, Immunohistochemistry, Fluorescence, Control, Software, Staining, Imaging