Journal:

Article Title: Nucleotide Exchange Factor ECT2 Interacts with the Polarity Protein Complex Par6/Par3/Protein Kinase C? (PKC?) and Regulates PKC? Activity

doi: 10.1128/MCB.24.15.6665-6675.2004

Figure Lengend Snippet: Par6 and ECT2 activate Cdc42 and PKCζ. (A) (Left panel) HeLa cells transfected with Myc-ECT2-ΔN5 together with Flag-Par6C vector or vector alone were lysed, and GTP-bound Cdc42 was pulled down with GST-PBD beads. The Western blot was carried out with anti-Cdc42 antibody. (Right panel) Similar experiments were performed in MDCK cells except that Flag-ECT2-ΔN5 was used. Accumulation of GTP-bound Rac1 was also estimated (lower panel). Similar results were obtained in three independent experiments. (B) Silencing of ECT2 reduces GTP-bound Cdc42. HeLa cells were treated with ECT2 siRNA or control luciferase siRNA (co) for 5 h and then transfected with Flag-Par6C. Cells were lysed 20 h after transfection, and GTP-bound Cdc42 was pulled down with GST-PBD-Sepharose beads. Western blots were carried out with anti-Cdc42, anti-ECT2, or anti-Flag antibody. These results were reproduced in three independent experiments. (C) ECT2 stimulates PKCζ activity. Cos7 cells transfected with the HA-tagged PKCζ expression vector in combination with the ECT2 and Flag-Par6C vectors or vector alone were lysed, and HA-PKCζ was immunoprecipitated with anti-HA antibody. PKCζ activity was estimated by in vitro kinase assays with myelin basic protein as the substrate, followed by autoradiography (upper panel). The level of HA-PKCζ was detected by immunoblotting with anti-HA antibody (lower panel). X-ray-films were scanned, and signals were quantitated with NIH Image software. Data are representative of three independent experiments.

Article Snippet: GTP-bound Cdc42 was estimated by the Cdc42 activation kit (Cytoskeleton Inc.).

Techniques: Transfection, Plasmid Preparation, Western Blot, Luciferase, Activity Assay, Expressing, Immunoprecipitation, In Vitro, Autoradiography, Software

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: Regulation of CDC42 activity by NDRG1 in CRC cells. A) Immunoblotting for total protein level or activated form of indicated Rho GTPase in NDRG1-modified HCT116 and RKO cells. Results are representative of at least three biological repeats, and the values in histograms are represented by mean ± S.D.; *P value <0.05, **P value <0.01, relative to the respective control cells. B) Confocal images were taken to show immunofluorescence staining of active-CDC42 (red) accompanied by the cell nucleus (blue) stained by DAPI in NDRG1 overexpression and NDRG1 knockdown HCT116 and RKO cells relative to the control cells, respectively. Fluorescence quantification was performed by comparing the integrated optical density (IOD)/area value of active-CDC42 to the IOD/area value of the nucleus (DAPI) in the same image. Results are representative of three to five images from different visual fields, and the histogram values are mean ±S.D. *P value <0.05, ***P<0.001, relative to the respective control cells. Scale bars: 25 µm.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Activity Assay, Western Blot, Modification, Control, Immunofluorescence, Staining, Over Expression, Knockdown, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: Inhibition of CDC42 prevents NDRG1 loss induced CRC cell filopodial protrusion formation through suppression of PAK1/Cofilin signaling. A) Immunoblotting analysis of the expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. B) Knockdown of CDC42 in HCT116 (left) and RKO (right) cells confirmed with immunoblotting analysis. Pool, combined siCDC42 sequences. C) Expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. D) Confocal images were taken to show immunofluorescence staining of MYO10 (green) and rhodamine-phalloidin (red) accompanied by the cell nucleus (blue) in colorectal cancer cells. Quantification of the MYO10-associated filopodial protrusions density and length is represented as mean ± S.D.; results are representative of 3-5 images from different visual fields, n>50 cells. *P value <0.05, **P value <0.01, ***P < 0.001, relative to the sh-Con/si-Con groups. # P value <0.05, ## P value <0.01, ### P < 0.001, relative to the sh-NDRG1/si-Con groups.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Inhibition, Western Blot, Expressing, Phospho-proteomics, Knockdown, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: NDRG1 suppresses CDC42 activity by stabilizing the RhoGDIα-CDC42 binding. A) The STRING network view of interactive proteins of CDC42 in humans. Gray lines between the nodes indicate various types of interaction evidence. B) Co-immunoprecipitation to examine the interaction of RhoGDIα and CDC42 in both HCT116 and RKO cell lines. C) Immunoblotting assay to evaluate the influence of NDRG1 modification on RhoGDIα expression in indicated cells. GAPDH was used as loading control. D) Double stained confocal immunofluorescence assay and co-localization analysis to confirm the interaction of RhoGDIα and CDC42 in indicated cells (red: CDC42, green: RhoGDIα, blue: DAPI, scale bar: 20 µm). Co-localization analysis on wide-field merged images was performed via Leica Application Suite X. Results are representative of five images from different visual fields.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Activity Assay, Binding Assay, Immunoprecipitation, Western Blot, Modification, Expressing, Control, Staining, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: Silence of NDRG1 promotes the peritoneal metastasis and correlates with upregulated CDC42 GTP expression. A) Peritoneal metastasis of CRC cells in BALB/c nude mice. Tumors in two groups were measured in situ and assessed by bioluminescence imaging in the fourth week. B) Statistical analysis of the bioluminescence in peritoneal foci of both groups. Results are shown as mean ± S.D. C) Tumors in two groups are demonstrated after laparotomy with hematoxylin-eosin staining of peritoneal foci on the lower panel. Scale bars are as indicated. D) Immunofluorescence staining of NDRG1 (left) or CDC42 GTP (right) accompanied by the cell nucleus stained by DAPI in peritoneal foci derived from sh-NDRG1 and control groups. Results are representative of 3-5 images from different visual fields and the histogram values are mean ± S.D.; *P value <0.05, ***P< 0.001, relative to the respective control groups. Scale bar: 50 µm.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Expressing, In Situ, Imaging, Staining, Immunofluorescence, Derivative Assay, Control

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: CDC42 GTP is frequently upregulated in CRC tissues and correlated with NDRG1 expression and clinicopathological parameters. A) IHC staining of NDRG1 and active CDC42 expression in tumor and adjacent tissues in microarray. Magnification on the right with a scale bar of 100 µm. B) Heatmap illustrating different clinicopathological parameters between CDC42 GTP -high and -low-expression tumors of the 86 cases. Statistical significance was analyzed by the χ 2 test. P values are as indicated.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Expressing, Immunohistochemistry, Microarray

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: Schematic diagram for the mechanism of NDRG1's regulation of CDC42/PAK1/Cofilin axis as a switch that modulates actin cytoskeleton rearrangement in human colorectal cancer invasion by stabilizing the RhoGDIα-CDC42 binding.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Binding Assay

Journal: Cell stem cell

Article Title: A FAK-YAP-mTOR signaling axis regulates stem cell-based tissue renewal in mice

doi: 10.1016/j.stem.2017.03.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-CDC42-GTP , NewEast Biosciences , Cat# 26905, RRID:AB_1961759.

Techniques: Plasmid Preparation, Recombinant, In Situ, Microarray, Sequencing, Software

Journal: Cell stem cell

Article Title: A FAK-YAP-mTOR signaling axis regulates stem cell-based tissue renewal in mice

doi: 10.1016/j.stem.2017.03.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-CDC42-GTP , NewEast Biosciences , Cat# 26905, RRID:AB_1961759.

Techniques: Plasmid Preparation, Recombinant, In Situ, Microarray, Sequencing, Software