Journal: FEBS Open Bio

Article Title: ASPM promotes hepatocellular carcinoma progression by activating Wnt/β‐catenin signaling through antagonizing autophagy‐mediated Dvl2 degradation

doi: 10.1002/2211-5463.13278

Figure Lengend Snippet: ASPM activates Wnt/β‐catenin signaling by interacting with Dvl2 in HCC cells. (A) Luciferase activity of TOP/FOPFlash in control or ASPM‐KD SMMC7721 and HepG2 cells. Data are shown as means ± SD ( n = 3 in each assay, Student's t ‐test). * P ≤ 0.05 vs. control shRNA. (B) Western blot analysis of the protein expression of Wnt/β‐catenin signaling‐related genes, including GSK‐3β, Axin, Dvl‐2, β‐catenin, TCF4, LEF1 and c‐Myc in control or ASPM‐KD SMMC7721 and HepG2 cells. (C) Reciprocal Co‐IP analysis between ASPM and Dvl2 in control or ASPM‐KD SMMC7721 (top panel) and HepG2 cells (bottom panel). (D) Immunofluorescence analysis of colocalization of ASPM (green) and Dvl‐2 (red) in SMMC7721 and HepG2 cells. Scale bars: 10 μm. IP, immunoprecipitation; WB, western blotting.

Article Snippet: After blocking with 0.5% BSA, the cells were incubated with rabbit polyclonal antibody ASPM (1 : 50 dilution, SC‐98903; Santa Cruz) and mouse monoclonal antibody Dvl‐2 (1 : 50 dilution, sc‐390303; Santa Cruz) at 4 °C overnight and then with fluorescence secondary antibodies (Alexa Fluor 594, SA00006‐3, 1 : 200 dilution; FITC, SA00003‐2, 1 : 200 dilution; Proteintech) at room temperature for 2 h. The nuclei were stained with DAPI for 2 min and washed with PBS.

Techniques: Luciferase, Activity Assay, Control, shRNA, Western Blot, Expressing, Co-Immunoprecipitation Assay, Immunofluorescence, Immunoprecipitation

Journal: FEBS Open Bio

Article Title: ASPM promotes hepatocellular carcinoma progression by activating Wnt/β‐catenin signaling through antagonizing autophagy‐mediated Dvl2 degradation

doi: 10.1002/2211-5463.13278

Figure Lengend Snippet: Overexpression of Dvl2 rescues the phenotypes of ASPM‐KD HCC cells. (A) Control or shASPM SMMC7721 and HepG2 cells were transfected with empty vector or Dvl‐2 expression vector, after which β‐catenin, Dvl‐2 and c‐Myc were detected by western blot. (B) Proliferation curve of control or shASPM SMMC7721 and HepG2 cells after transient transfections with empty vector or Dvl2 expression vector. Data are shown as means ± SD ( n = 3 in each assay, Student's t ‐test). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. shASPM + Vector. (C) Representative images of scratch wound assays of control or shASPM SMMC7721 and HepG2 cells after transient transfections with empty vector or Dvl‐2 expression vector. Scale bars: 100 μm. Data are shown as means ± SD ( n = 3 in each assay, Student's t ‐test). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. shASPM + Vector.

Article Snippet: After blocking with 0.5% BSA, the cells were incubated with rabbit polyclonal antibody ASPM (1 : 50 dilution, SC‐98903; Santa Cruz) and mouse monoclonal antibody Dvl‐2 (1 : 50 dilution, sc‐390303; Santa Cruz) at 4 °C overnight and then with fluorescence secondary antibodies (Alexa Fluor 594, SA00006‐3, 1 : 200 dilution; FITC, SA00003‐2, 1 : 200 dilution; Proteintech) at room temperature for 2 h. The nuclei were stained with DAPI for 2 min and washed with PBS.

Techniques: Over Expression, Control, Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: FEBS Open Bio

Article Title: ASPM promotes hepatocellular carcinoma progression by activating Wnt/β‐catenin signaling through antagonizing autophagy‐mediated Dvl2 degradation

doi: 10.1002/2211-5463.13278

Figure Lengend Snippet: ASPM stabilizes Dvl2 via antagonizing the autophagy system. (A) The transcript levels of Dvl2 and CTNNB1 in control or shASPM SMMC7721 and HepG2 cells measured by qRT‐PCR. Data are means ± SD ( n = 3 in each assay, Student's t ‐test). (B) Western blot analysis of Dvl2 protein levels in control or shASPM SMMC7721 and HepG2 cells at 0, 4 and 6 h after treatment with autophagy inhibitor 3‐MA (10 mM), respectively. (C) Western blot analysis of LC3II and p62 protein levels in control or shASPM SMMC7721 and HepG2 cells. (D) Co‐IP analysis between Dvl2 and LC3II in control or shASPM SMMC7721 and HepG2 cells. (E) The proposed model illustrates how ASPM promotes HCC progression. Left panel: without ASPM, Dvl2 is degraded by the autophagic lysosomal pathway, and β‐catenin is destroyed by destruction complex, which prevents the accumulation of β‐catenin protein in the cytoplasm and thereby the inactivation of the Wnt/β‐catenin pathway. Right panel: when ASPM binds with Dvl2, it stabilizes Dvl2 protein via antagonizing the autophagy system, thereby resulting in the translocation of β‐catenin into the nucleus and augmenting Wnt/β‐catenin signaling.

Article Snippet: After blocking with 0.5% BSA, the cells were incubated with rabbit polyclonal antibody ASPM (1 : 50 dilution, SC‐98903; Santa Cruz) and mouse monoclonal antibody Dvl‐2 (1 : 50 dilution, sc‐390303; Santa Cruz) at 4 °C overnight and then with fluorescence secondary antibodies (Alexa Fluor 594, SA00006‐3, 1 : 200 dilution; FITC, SA00003‐2, 1 : 200 dilution; Proteintech) at room temperature for 2 h. The nuclei were stained with DAPI for 2 min and washed with PBS.

Techniques: Control, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Translocation Assay

Journal: Science (New York, N.Y.)

Article Title: RIPK4 phosphorylates Dishevelled proteins to regulate canonical Wnt signaling

doi: 10.1126/science.1232253

Figure Lengend Snippet: A. HEK293T cells were transfected with control (Ctrl) or RIPK4 siRNAs for 72 h and then given vehicle or 200 ng/mL Wnt3a for 30 min. B. RIPK4-Myc and DVL2-FLAG were expressed with rabbit reticulocyte and wheat germ in vitro translation systems, respectively. The proteins were mixed and immunoprecipitated with anti-FLAG beads. C. HEK293T cells treated with 200 ng/mL Wnt3a. D. In vitro kinase assays using the RIPK4 kinase domain (wild-type or K51R mutant) and FLAG-tagged DEP or PDZ domains of DVL2. E. HEK293T cells transfected with control or RIPK4 siRNA for 72 h, and then given vehicle or 200 ng/mL Wnt3a for 10 min. Non-specific bands (*). F. Dvl-null MEFs reconstituted with empty vector or DVL2 (wild-type or mutant S298A S480A) and treated with Wnt3a. G. Immunofluorescence microscopy of HeLa cells transfected with RIPK4-GFP and DVL2-FLAG for 36 h. Cells containing DVL2 puncta (signalosome) were enumerated by counting 250 cells per condition. Scale bars: 10 μm.

Article Snippet: DVL2 phospho-Ser298 and DVL2 phospho-Ser480 rabbit polyclonal antibodies were generated by Prosci, and monoclonal antibodies were then derived by Epitomics and Genentech.

Techniques: Transfection, Control, In Vitro, Immunoprecipitation, Mutagenesis, Plasmid Preparation, Immunofluorescence, Microscopy

Journal: Molecular Neurobiology

Article Title: GIV/Girdin Modulation of Microglial Activation in Ischemic Stroke: Impact of FTO-Mediated m6A Modification

doi: 10.1007/s12035-024-04604-8

Figure Lengend Snippet: GIV interacts with DVL2 and activates Wnt/β-catenin signaling pathway. A Protein-protein interaction network analysis (PPI). B Structure-based protein interaction interface analysis between GIV and DVL2. C Representative images of Western blot after GIV overexpression. D Representative images of western blot after GIV knockdown. E Representative images of immunofluorescent staining. F DVL2, GIV, and IgG antibody, THP-1 lysates were immunoprecipitated using DVL2 antibody and then analyzed by Western blot using the indicated antibodies. G Half-life of DVL2 in BV-2 cells with Vector or overexpression of GIV, treated with cycloheximide (CHX) at the indicated times and analyzed by Western blot ( n = 3, ** P < 0.01, scale bar = 20 μm)

Article Snippet: The following antibodies were used in this study: FTO (D2V1I) Rabbit mAb (#45980; CST), anti-GIV antibody (ab179481; Abcam), N6-methyladenosine(m6A) (D9D9W) rabbit mAb (#56593; CST), β-actin mouse mAb (#3700; CST), GAPDH monoclonal antibody (60004-1-Ig; Proteintech; Wuhan, China), iNOS rabbit pAb (A14031; ABclonal; Wuhan, China), arginase 1 (ARG1) rabbit pAb (A1847; ABclonal), CD16 polyclonal antibody (16559-1-AP; Proteintech), CD206 polyclonal antibody (18704-1-AP; Proteintech), Iba1 polyclonal antibody (10904-1-AP; Proteintech), GSK-3β rabbit mAb (#12456; CST; USA), β-catenin rabbit mAb (#8480; CST; USA), AXIN2 polyclonal antibody (20540-1-AP; Proteintech), cyclin D1 rabbit mAb (#55506; CST; USA), DVL2 polyclonal antibody (12037-1-AP; Proteintech).

Techniques: Western Blot, Over Expression, Knockdown, Staining, Immunoprecipitation, Plasmid Preparation