active rac1 pulldown and detection kit  (Thermo Fisher)

Journal: Frontiers in Oncology

Article Title: RAC1 Involves in the Radioresistance by Mediating Epithelial-Mesenchymal Transition in Lung Cancer

doi: 10.3389/fonc.2020.00649

Figure Lengend Snippet: RAC1 regulates cell proliferation in vivo and in vitro in lung cancer cells. (A) The successful overexpression/downregulation of RAC1 protein in A549 and PC9 cells was detected by immunoblotting. (B) Overexpression of RAC1 promoted A549 and PC9 cell clone formation capability and silence of RAC1 inhibited cell clone formation capability, which were analyzed by colony formation assay and crystal violet staining after 14 days, clone numbers were quantified. (C) The effect of RAC1 expression onA549 and PC9 cell proliferation was assessed by the CCK-8 cell growth assay. A549 and PC9 cells transfected with CMV-RAC1 or CMV-sh-RAC1 plasmid, Vector cells transfected with CMV plasmid or CMV-sh-control plasmid. (D–F) RAC1 expression increased tumor growth in vivo . The subcutaneous transplantation tumor model in nude BALB/c mice was established using A549 cells that were stably transduced with RAC1 and sh-RAC1. The tumor size was measured at pointed time and tumor weight was measured. Data are expressed as the mean ± SD of different groups of cells from three separate experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Rac1 pulldown assay was performed using commercial kit (#8815, CST) as described recently ( ).

Techniques: In Vivo, In Vitro, Over Expression, Western Blot, Colony Assay, Staining, Expressing, CCK-8 Assay, Growth Assay, Transfection, Plasmid Preparation, Control, Transplantation Assay, Stable Transfection, Transduction

Journal: Frontiers in Oncology

Article Title: RAC1 Involves in the Radioresistance by Mediating Epithelial-Mesenchymal Transition in Lung Cancer

doi: 10.3389/fonc.2020.00649

Figure Lengend Snippet: Increased RAC1 expression by irradiation is closely related to EMT markers expression in lung cancer cells. (A,B) mRNA and protein levels of RAC1, Vimentin and E-cadherin in A549 cells exposure to 0, 2, 4, 6, 8 Gy dose of irradiation. (C) Rac1 expression and activity was monitored in A549 and PC9 cells exposure to 6 Gy dose of irradiation. (D) The protein levels of RAC1, PI3K, p-AKT, AKT in A549 and PC9 cells exposure to 6 Gy dose of irradiation. (E) The protein levels of RAC1, PI3K, p-AKT, AKT in A549 and PC9 cells exposure to 6 Gy dose of irradiation was monitored in response to LY294002 treatment. Data are expressed as the mean ± SD of different groups of cells from three separate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rac1 pulldown assay was performed using commercial kit (#8815, CST) as described recently ( ).

Techniques: Expressing, Irradiation, Activity Assay

Journal: Frontiers in Oncology

Article Title: RAC1 Involves in the Radioresistance by Mediating Epithelial-Mesenchymal Transition in Lung Cancer

doi: 10.3389/fonc.2020.00649

Figure Lengend Snippet: RAC1 inhibition increased the radio-sensitivity for lung cancer cells. (A) RAC1-overexpressing A549 and PC9 cells exposure to 0, 2, 4, 6, 8 Gy dose of irradiation promoted cellular radioresistance, and RAC1 silenced A549 and PC9 cells exposure to 0, 2, 4, 6, 8 Gy dose of irradiation attenuated cellular radioresistance under as determined by the clonogenic assay. (Down panel) Dose-survival curve derived from the results of clonogenic assay for A549 and PC9 cells. SF = colonies/(inoculation cells × survival rate). SF represents the sensitivity of cells to RAC1 expression exposed to IR. These numbers (0.1, 0.5, 1.5, 5, and 10 k) represent the number of seeded cells (100, 500, 1,500, 5,000, and 10,000). (B) Cell proliferation ability of A549 and PC9 cells within indicated group were detected by CCK-8 assay. Data are expressed as the mean ± SD of different groups of cells from three separate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rac1 pulldown assay was performed using commercial kit (#8815, CST) as described recently ( ).

Techniques: Inhibition, Irradiation, Clonogenic Assay, Derivative Assay, Expressing, CCK-8 Assay

Journal: Frontiers in Oncology

Article Title: RAC1 Involves in the Radioresistance by Mediating Epithelial-Mesenchymal Transition in Lung Cancer

doi: 10.3389/fonc.2020.00649

Figure Lengend Snippet: The effects of RAC1 expression on irradiation-induced migration and invasion in vitro . A549 and PC9 cells were exposed to 6 Gy doses of IR in the presence or absence of RAC1 expression. (A) The influence of RAC1 expression on lung cancer cell migration was determined by wound healing assay in A549 and PC9 cells. Scale bar is 100 μm. (B) The effects of RAC1 expression on cell migration detected by Transwell assay, scale bar is 50 μm. (C) The effects of RAC1 expression on cell invasion detected by Transwell assay, scale bar is 50 μm. Data are expressed as the mean ± SD of different groups of cells from three separate experiments. ** P < 0.01, *** P < 0.001.

Article Snippet: Rac1 pulldown assay was performed using commercial kit (#8815, CST) as described recently ( ).

Techniques: Expressing, Irradiation, Migration, In Vitro, Wound Healing Assay, Transwell Assay

Journal: Frontiers in Oncology

Article Title: RAC1 Involves in the Radioresistance by Mediating Epithelial-Mesenchymal Transition in Lung Cancer

doi: 10.3389/fonc.2020.00649

Figure Lengend Snippet: IR induced EMT progression by activating Rac1 signaling pathway in lung cancer cells. A549 and PC9 cells were exposed to 6 Gy radiation in the overexpression and the absence of Rac1 expression. The expression of EMT-related markers, Rac1 expression and activity, and Rac1 signaling pathway related markers (indicated in the figure) was monitored in response to IR treatment. Data are expressed as the mean ± SD of different groups of cells from three separate experiments. * P <0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rac1 pulldown assay was performed using commercial kit (#8815, CST) as described recently ( ).

Techniques: Over Expression, Expressing, Activity Assay

Journal: Frontiers in Oncology

Article Title: RAC1 Involves in the Radioresistance by Mediating Epithelial-Mesenchymal Transition in Lung Cancer

doi: 10.3389/fonc.2020.00649

Figure Lengend Snippet: The effects of RAC1 expression in radiotherapy in vivo . (A) Representative photo of residual tumor of after 3*5 Gy dose of irradiation. (B) The time course of growth of Vector, RAC1, sh-control and sh-RAC1 xenograft tumors with or without IR treatment. (C) IHC staining showed an elevated Ki67 expression and upregulation of RAC1 in RAC1 overexpression xenograft tumor, which represents the radioresistant process in RAC1 overexpressing cells in vivo . Right panel showed a decreased Ki67 expression and downregulation of RAC1 in silencing RAC1 xenograft tumor, which represents the radiosensitivity process in sh-RAC1 cells in vivo . Down panel showed the quantification of scoring of immunostaining in RAC1 overexpression/silencing xenograft. Scale bar 100 μm. Data are expressed as the mean ± SD of different groups of cells from three separate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rac1 pulldown assay was performed using commercial kit (#8815, CST) as described recently ( ).

Techniques: Expressing, In Vivo, Irradiation, Plasmid Preparation, Control, Immunohistochemistry, Over Expression, Immunostaining