Journal: Cells

Article Title: NEK2 Phosphorylates RhoGDI1 to Promote Cell Proliferation, Migration and Invasion Through the Activation of RhoA and Rac1 in Colon Cancer Cells

doi: 10.3390/cells13242072

Figure Lengend Snippet: NEK2 interacts with RhoGDI1 but not RhoGDI2. ( A ) HeLa cells were co-transfected with HA-tagged NEK2 and Flag-tagged RhoGDI1 or RhoGDI2. Cell lysates were subjected to immunoprecipitation using HA antibody, followed by Western blotting with HA and Flag antibodies. ( B ) GST pull-down assay was conducted using recombinant His-tagged NEK2 and GST-tagged RhoGDI1 or RhoGDI2. ( C ) Western blot analysis was performed to assess NEK2 and RhoGDI1 expression in human colon cancer cell lines. ( D ) Cell lysates from HT-29 and HCT116 were immunoprecipitated with either IgG or RhoGDI1 antibodies, followed by Western blot analysis using indicated antibodies.

Article Snippet: For GST pull-down assay, The GSH-Beads (GE Healthcare, Chicago, IL, USA, 17-0756-01) were reacted with recombinant RhoGDI1 (Cytoskeleton, Denver, CO, GDI01) and RhoGDI2 (Novusbio, E Easter Ave, Centennial, CO, USA, NBP1-99054) proteins overnight at 4 °C and then mixed with active His-NEK2 (Thermo Fisher Scientific, Waltham, MA, USA, PV3360) overnight at 4 °C.

Techniques: Transfection, Immunoprecipitation, Western Blot, Pull Down Assay, Recombinant, Expressing

Journal: Cells

Article Title: NEK2 Phosphorylates RhoGDI1 to Promote Cell Proliferation, Migration and Invasion Through the Activation of RhoA and Rac1 in Colon Cancer Cells

doi: 10.3390/cells13242072

Figure Lengend Snippet: The requirement of the RhoGDI1 aa 112–134 region for its interaction with NEK2. ( A ) Schematic diagram of RhoGDI1 WT and 6 truncated fragments. ( B , C ) Purified GST-tagged RhoGDI1 WT or truncation fragments along with recombinant His-NEK2 were subjected to His pull-down assay. His pull-down samples were analyzed by WB using NEK2 and GST antibodies. ( D ) HCT116 cells were transfected with the mock vector or GFP-tagged RhoGDI1 aa 112–134 fragment. Cell lysates were immunoprecipitated with either IgG or RhoGDI1 antibodies, followed by Western blot analysis using indicated antibodies (left). Relative band intensities (NEK2/RhoGDI1) were quantified using Image J and shown as a graph (right). Quantitative data represent the mean ± S.D. ( n = 3). ** p < 0.01.

Article Snippet: For GST pull-down assay, The GSH-Beads (GE Healthcare, Chicago, IL, USA, 17-0756-01) were reacted with recombinant RhoGDI1 (Cytoskeleton, Denver, CO, GDI01) and RhoGDI2 (Novusbio, E Easter Ave, Centennial, CO, USA, NBP1-99054) proteins overnight at 4 °C and then mixed with active His-NEK2 (Thermo Fisher Scientific, Waltham, MA, USA, PV3360) overnight at 4 °C.

Techniques: Purification, Recombinant, Pull Down Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Cells

Article Title: NEK2 Phosphorylates RhoGDI1 to Promote Cell Proliferation, Migration and Invasion Through the Activation of RhoA and Rac1 in Colon Cancer Cells

doi: 10.3390/cells13242072

Figure Lengend Snippet: NEK2 phosphorylates RhoGDI1 at Ser174. ( A ) Purified His-RhoGDI1 were subjected to in vitro kinase assay with recombinant active NEK2 (+) or without NEK2 (−). Samples were then analyzed by WB using indicated antibodies. ( B ) Purified His-RhoGDI1 WT and substituted mutants (S34A, S96A, S101A, S174A and T7/91A) were subjected to in vitro kinase assay with recombinant active NEK2 (+) or without NEK2 (−), followed by WB analysis using indicated antibodies. The arrow indicates phosphorylated RhoGDI1. ( C ) DLD-1 cells were stably transfected with HA-NEK2. ( C , D ) DLD-1 and HCT116 cells were incubated with 50 nM of NCL 00017509 (NEK2 inhibitor) in serum-free media for 24 h, followed by WB analysis using indicated antibodies (left). Relative band intensities (p-RhoGDI1/RhoGDI1) were quantified using Image J and shown as a graph (right). ( E ) HCT116 cells stably expressing control shRNA or two NEK2 shRNAs were incubated in serum-free media for 24 h. Cell lysates were subjected to WB analysis using indicated antibodies (upper). Relative band intensities (p-RhoGDI1/RhoGDI1) (lower). ( F ) DLD-1 cells stably expressing mock or HA-NEK2 were transiently transfected with mock or GFP-RhoGDI1 aa 112–134 expressing vector. Cells were incubated in serum-free media for 24 h. Cell lysates were analyzed by WB using indicated antibodies (upper). Relative band intensities (p-RhoGDI1/RhoGDI1) (lower). ( G ) DLD-1 cells stably expressing mock or HA-NEK2 were incubated in serum-free media for 24 h. IP analysis was performed with DLD-1 cell lysates and a RhoGDI1 antibody, followed by WB analysis using indicated antibodies (upper). Relative band intensities (14-3-3 tau/RhoGDI1) (lower). Quantitative data represent the mean ± S.D. ( n = 3). * p < 0.05; ** p < 0.01.

Article Snippet: For GST pull-down assay, The GSH-Beads (GE Healthcare, Chicago, IL, USA, 17-0756-01) were reacted with recombinant RhoGDI1 (Cytoskeleton, Denver, CO, GDI01) and RhoGDI2 (Novusbio, E Easter Ave, Centennial, CO, USA, NBP1-99054) proteins overnight at 4 °C and then mixed with active His-NEK2 (Thermo Fisher Scientific, Waltham, MA, USA, PV3360) overnight at 4 °C.

Techniques: Purification, In Vitro, Kinase Assay, Recombinant, Stable Transfection, Transfection, Incubation, Expressing, Control, shRNA, Plasmid Preparation

Journal: Cells

Article Title: NEK2 Phosphorylates RhoGDI1 to Promote Cell Proliferation, Migration and Invasion Through the Activation of RhoA and Rac1 in Colon Cancer Cells

doi: 10.3390/cells13242072

Figure Lengend Snippet: NEK2 facilitates the activation of RhoA and Rac1 by interaction with RhoGDI1. ( A ) DLD-1 cells stably expressing mock or HA-NEK2 were incubated in serum-free media for 24 h. A pull-down assay was performed to assess the levels of active RhoA and Rac1/Cdc42, as described in the Materials and Methods Section (left). ( B ) DLD-1 cells stably expressing mock or HA-NEK2 were treated with 50 nM of NCL 00017509 in serum-free media for 24 h, followed by pull-down assay and WB analysis (left). ( C ) HCT116 cells stably expressing control shRNA or two NEK2 shRNAs were incubated in serum-free media for 24 h and subjected to pull-down assay and WB analysis (left). ( D ) HA-NEK2 expressing DLD-1 cells were stably transfected with mock or mCherry-RhoGDI1 aa 112–134 expressing vector. Cells were incubated in serum-free media for 24 h, followed by pull-down assay and WB analysis (left). Relative band intensities (GTP-RhoA/total RhoA) were measured by Image J and shown as a graph (right). Quantitative data represent the mean ± S.D. ( n = 3). * p < 0.05; ** p < 0.01.

Article Snippet: For GST pull-down assay, The GSH-Beads (GE Healthcare, Chicago, IL, USA, 17-0756-01) were reacted with recombinant RhoGDI1 (Cytoskeleton, Denver, CO, GDI01) and RhoGDI2 (Novusbio, E Easter Ave, Centennial, CO, USA, NBP1-99054) proteins overnight at 4 °C and then mixed with active His-NEK2 (Thermo Fisher Scientific, Waltham, MA, USA, PV3360) overnight at 4 °C.

Techniques: Activation Assay, Stable Transfection, Expressing, Incubation, Pull Down Assay, Control, shRNA, Transfection, Plasmid Preparation

Journal: Cells

Article Title: NEK2 Phosphorylates RhoGDI1 to Promote Cell Proliferation, Migration and Invasion Through the Activation of RhoA and Rac1 in Colon Cancer Cells

doi: 10.3390/cells13242072

Figure Lengend Snippet: NEK2 promotes migration and invasion of colon cancer cells by phosphorylating RhoGDI1. ( A – C ) HCT116 cells were incubated with 50 nM of NCL 00017509, NEK2 inhibitor. ( A ) The viability of treated cells was assessed using a WST-8 assay. The graph represents the relative percentages of proliferating cells compared to untreated control. ( B ) Cell migration was evaluated using wound-healing assay at indicated time point. Representative images of migrating cells obtained at 48 h after wound formation (left). Scale bar = 200 μm. The migration was quantified by calculating the cell-covered area using Image J (right). ( C ) Cells were incubated in serum-free media for 24 h and subjected to transwell invasion assay. Representative images (100×) of invading cells are shown on the left, and the relative percentages of invasion are presented on the right. ( D , E ) DLD-1 cells stably expressing mock or HA-NEK2 were transiently transfected with Flag-RhoGDI1 WT or Flag-RhoGDI1 S174A. ( D ) Cell migration was evaluated using wound-healing assay at indicated time point. Representative images of migrating cells are shown on the left, and the percentage of wound closure is depicted on the right. Scale bar = 200 μm. ( E ) Cells were incubated in serum-free media for 24 h and subjected to transwell invasion assay. Migrating or invading cells were shown in representative images (left) or the relative percentages of invasion (right). Quantitative data represent the mean ± S.D. ( n = 3). ** p < 0.01; NS, non-significant.

Article Snippet: For GST pull-down assay, The GSH-Beads (GE Healthcare, Chicago, IL, USA, 17-0756-01) were reacted with recombinant RhoGDI1 (Cytoskeleton, Denver, CO, GDI01) and RhoGDI2 (Novusbio, E Easter Ave, Centennial, CO, USA, NBP1-99054) proteins overnight at 4 °C and then mixed with active His-NEK2 (Thermo Fisher Scientific, Waltham, MA, USA, PV3360) overnight at 4 °C.

Techniques: Migration, Incubation, Control, Wound Healing Assay, Transwell Invasion Assay, Stable Transfection, Expressing, Transfection

Journal: Cells

Article Title: NEK2 Phosphorylates RhoGDI1 to Promote Cell Proliferation, Migration and Invasion Through the Activation of RhoA and Rac1 in Colon Cancer Cells

doi: 10.3390/cells13242072

Figure Lengend Snippet: Interaction of NEK2 with aa 112–134 on RhoGDI1 is crucial for promoting proliferation, migration and invasion of colon cancer cells. DLD-1 cells expressing mock or HA-NEK2 were stably transfected with mCherry or mCherry-RhoGDI1 aa 112–134. ( A ) Cells were subjected to WST-8 assay. The graph represents the relative percentages of proliferating cells. ( B ) Migration of indicated cells was evaluated by wound-healing assay at each time point. Representative images of migrating cells obtained at 24 h after wound formation (left). Scale bar = 200 μm. The migration was quantified by calculating the cell-covered area using Image J (right). ( C ) Cells were incubated in serum-free media for 24 h and then subjected to transwell invasion assay. Representative images (100×) of invading cells are displayed on the left, and the relative percentages of invasion are quantified on the right. Quantitative data represent the mean ± S.D. ( n = 3). * p < 0.05; ** p < 0.01.

Article Snippet: For GST pull-down assay, The GSH-Beads (GE Healthcare, Chicago, IL, USA, 17-0756-01) were reacted with recombinant RhoGDI1 (Cytoskeleton, Denver, CO, GDI01) and RhoGDI2 (Novusbio, E Easter Ave, Centennial, CO, USA, NBP1-99054) proteins overnight at 4 °C and then mixed with active His-NEK2 (Thermo Fisher Scientific, Waltham, MA, USA, PV3360) overnight at 4 °C.

Techniques: Migration, Expressing, Stable Transfection, Transfection, Wound Healing Assay, Incubation, Transwell Invasion Assay

Journal: Cells

Article Title: NEK2 Phosphorylates RhoGDI1 to Promote Cell Proliferation, Migration and Invasion Through the Activation of RhoA and Rac1 in Colon Cancer Cells

doi: 10.3390/cells13242072

Figure Lengend Snippet: NEK2 promotes tumor growth and metastasis of colon cancer through its association with RhoGDI1. ( A – D ) DLD-1 cells expressing either mCherry or mCherry-RhoGDI1 aa 112–134 with or without HA-NEK2 were subcutaneously inoculated into the mice (5 × 10 6 /mouse). ( A ) Representative image of the tumors from each group of mice. ( B ) Measurement of tumor weights. ( C ) Measurement of tumor volumes, following procedures described in the Materials and Methods Section. ( D ) Tumor sections of each mouse were stained with anti-Ki67 or anti-CD31 antibody to assess proliferation and angiogenesis, respectively. Scale bar = 500 μm. Insets display accumulation of Ki-67 or CD31. Scale bar = 100 µm. ( E , F ) Indicated DLD-1 were intravenously inoculated into the mice (2 × 10 6 /mouse). ( E ) Representative images of H&E-stained lung tissues of the mice injecting DLD-1 cell lines. Scale bar = 500 μm. ( F ) The number of metastatic nodules was counted per lung tissue and represented in the scatter plots. Quantitative data represent the mean ± S.D. ( n = 8). ** p < 0.01.

Article Snippet: For GST pull-down assay, The GSH-Beads (GE Healthcare, Chicago, IL, USA, 17-0756-01) were reacted with recombinant RhoGDI1 (Cytoskeleton, Denver, CO, GDI01) and RhoGDI2 (Novusbio, E Easter Ave, Centennial, CO, USA, NBP1-99054) proteins overnight at 4 °C and then mixed with active His-NEK2 (Thermo Fisher Scientific, Waltham, MA, USA, PV3360) overnight at 4 °C.

Techniques: Expressing, Staining