gst inhibitors  (Valiant Co Ltd)

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

Journal: Chemical Science

Article Title: High-efficiency enzymatic biodegradation of polypropylene-based melt-blown fabric debris †

doi: 10.1039/d5sc03097h

Figure Lengend Snippet: Characterization of morphology, particle size, degradation kinetics, and degradation efficiency during the degradation of MBFD induced by trypsin and GST. (A–D) Characterization of morphology and particle size during the degradation of MBFD induced by trypsin and GST. SEM images and the corresponding size variations under different conditions of treatment with (A) trypsin and (B) GST are shown. (C) Dynamic characterization of MBFD at different incubation times (0.1–4 days), including SEM images and particle size distribution. (D) SEM images and particle size distribution during the degradation of MBFD induced by GST in the presence of small-molecule inhibitors. (E–J) Monitoring the enzymatic depolymerization of MBFD. (E, F, H, and I) Degradation kinetics of MBFD under different conditions. The dynamic curves of MBFD (E and H) in water and (F and I) in wastewater (WW) samples. The degradation efficiency of MBFD treated with (G) trypsin and (J) GST in different ambient environments. Each filled symbol represents the mean ± SD ( n = 3).

Article Snippet: The inhibitors for GST (indomethacin, 99%) and trypsin (trypsin inhibitor soybean) were purchased from Rhawn (Shanghai, China) and Aladdin (Shanghai, China), respectively. l -γ-Glutamyl- S -(phenylmethyl)- l -cysteinyl-2-phenyl (TLK117) was purchased from MedChemExpress (New Jersey, USA). (3 S )-1-Chloro-3-tosylamido-7-amino-2-heptanone hydrochloride (TLCK) was obtained from Biolab (Beijing, China).

Techniques: Incubation