Journal: International Journal of Molecular Sciences

Article Title: Caveolin-1 Limits the Contribution of BKCa Channel to MCF-7 Breast Cancer Cell Proliferation and Invasion

doi: 10.3390/ijms151120706

Figure Lengend Snippet: Immunofluorescence and co-immunoprecipitation analysis of BKCa and caveolin-1 in human breast cancer MCF-7 cells. ( A ) MCF-7 cells were immunostained with the anti-BKCa (red) and anti-caveolin (green) antibodies. The nuclei were stained with DAPI (blue). The merged image revealed that BKCa and caveolin-1 are co-localised. Scale bar = 20 μm; and ( B ) Anti-BKCa antibody co-immunoprecipitated caveolin-1 from a total protein lysate prepared from MCF-7 cells. Reciprocally, the anti-caveolin antibody co-immunoprecipitated BKCa. Whole cell lysate was probed for input. Bead lanes contain the protein G conjugated sepharose beads used during the immunoprecipitation without the protein input. All the experiments were repeated three times.

Article Snippet: The proteins were detected with the following antibodies: Rabbit anti-BKCa polyclone antibody (1:600, Alomone, Jerusalem, Israel), rabbit anti-caveolin-1 polyclone antibody (1:1000, Abcam).The primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (1:15000, Abcam), and the signals were developed using an enhanced chemiluminescence detection kit (Thermo).

Techniques: Immunofluorescence, Immunoprecipitation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Caveolin-1 Limits the Contribution of BKCa Channel to MCF-7 Breast Cancer Cell Proliferation and Invasion

doi: 10.3390/ijms151120706

Figure Lengend Snippet: Caveolin-1 knockdown increased the surface expression and activity of BKCa channels in MCF7 Cells. ( A ) MCF-7 cells were transfected with 30 and 60 nM caveolin-1 siRNA (siCav-1) or scrambled siRNA (si Control) for 48 h. Caveolin-1, membrane BKCa (M-BKCa), total BKCa (T-BKCa) and β-actin protein expressions in the cells were analyzed by western blot; ( B ) MCF-7 cells were transfected with 30 and 60 nM BKCa siRNA (siBKCa) or scrambled siRNA (si Control) for 48 h. Caveolin-1, BKCa and β-actin protein expressions in the cells were analyzed by Western blotting; ( C ) Whole-cell K + currents in MCF-7 cells transfected with 30 nM siControl, siBKCa or siCav-1 for 48 h; ( D ) Group data of current-voltage relationships in MCF-7 cells treated with 30 nM siControl ( n = 10), siBKCa ( n = 9) or siCav-1 ( n = 8) for 48 h; ( E ) Representative traces of BKCa single-channel currents in cell-attached patches after the treatment of 30 nM siControl or siCav-1 for 48 h; ( F ) NPo (Po, open probability) was calculated in siControl or siCav-1 transfected MCF-7 cells. siCav-1 increased the NPo significantly; and ( G ) Unitary current amplitude (Am) in BKCa channels were shown against membrane potentials. No significant difference was observed between the two groups. All the experiments were repeated three times. ( * p < 0.05).

Article Snippet: The proteins were detected with the following antibodies: Rabbit anti-BKCa polyclone antibody (1:600, Alomone, Jerusalem, Israel), rabbit anti-caveolin-1 polyclone antibody (1:1000, Abcam).The primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (1:15000, Abcam), and the signals were developed using an enhanced chemiluminescence detection kit (Thermo).

Techniques: Knockdown, Expressing, Activity Assay, Transfection, Control, Membrane, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Caveolin-1 Limits the Contribution of BKCa Channel to MCF-7 Breast Cancer Cell Proliferation and Invasion

doi: 10.3390/ijms151120706

Figure Lengend Snippet: Caveolin-1 knockdown mediated activation of BKCa channel promotes proliferation and invasion in MCF-7 cells. MCF-7 cells were transfected with indicated amounts of siRNAs for 48 h. For MTT assays ( A – C ), cells were plated in 96-well plates at an initial density of 4000 cells/well and cultured for the indicated time; For invasion assay ( D – F ), cells were plated in 24-well plates at an initial density of 30,000 cells/well and cultured for 24 h. Representative photographs of tumor cells that invaded through a Matrigel-coated filter were taken and shown. The invasive cells were counted in 10 random fields. The experiment was repeated at least three times and results are expressed relative to the number of control. All the experiments were repeated three times. (* p < 0.05).

Article Snippet: The proteins were detected with the following antibodies: Rabbit anti-BKCa polyclone antibody (1:600, Alomone, Jerusalem, Israel), rabbit anti-caveolin-1 polyclone antibody (1:1000, Abcam).The primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (1:15000, Abcam), and the signals were developed using an enhanced chemiluminescence detection kit (Thermo).

Techniques: Knockdown, Activation Assay, Transfection, Cell Culture, Invasion Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Caveolin-1 Limits the Contribution of BKCa Channel to MCF-7 Breast Cancer Cell Proliferation and Invasion

doi: 10.3390/ijms151120706

Figure Lengend Snippet: Caveolin-1 overexpression inhibited the expression and activity of BKCa Channels in MCF7 Cells. ( A ) MCF-7 cells were transfected with 3 and 6 μg BKCa plasmid (pBKCa) or control vector for 48 h. Caveolin-1, membrane BKCa (M-BKCa), total BKCa (T-BKCa) and β-actin protein expressions in the cells were analyzed by Western blotting; ( B ) MCF-7 cells were transfected with 3 and 6 μg caveolin-1 plasmid (pCav-1) or control vector for 48 h. Caveolin-1, BKCa and β-actin protein expressions in the cells were analyzed by western blot; ( C ) Whole-cell K + currents in MCF-7 cells transfected with 3 μg pCav-1, pBKCa or vector for 48 h; ( D ) Group data of current-voltage relationships in MCF-7 cells treated with 3 μg pCav-1 ( n = 9), pBKCa ( n = 8) or vector ( n = 8) for 48 h; ( E ) Representative traces of BKCa single-channel currents in cell-attached patches after the treatment of 3 μg pCav-1 or vector for 48 h; ( F ) NPo (Po, open probability) was calculated in pCav-1 or vector transfected MCF-7 cells. pCav-1 decreased the NPo significantly ; and ( G ) Unitary current amplitude (Am) in BKCa channels were shown against membrane potentials. No significant difference could be observed between the two groups. All the experiments were repeated three times. (* p < 0.05).

Article Snippet: The proteins were detected with the following antibodies: Rabbit anti-BKCa polyclone antibody (1:600, Alomone, Jerusalem, Israel), rabbit anti-caveolin-1 polyclone antibody (1:1000, Abcam).The primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (1:15000, Abcam), and the signals were developed using an enhanced chemiluminescence detection kit (Thermo).

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

Journal: International Journal of Molecular Sciences

Article Title: Caveolin-1 Limits the Contribution of BKCa Channel to MCF-7 Breast Cancer Cell Proliferation and Invasion

doi: 10.3390/ijms151120706

Figure Lengend Snippet: Caveolin-1 upregulation suppressed proliferation and invasion via decreasing expression and function of BKCa channel in MCF7 cells. MCF-7 cells were transfected with indicated amounts of plasmids for 48 h. For MTT assays ( A – C ), cells were plated in 96-well plates at an initial density of 4000 cells/well and cultured for the indicated time. For invasion assay ( D – F ), cells were plated in 24-well plates at an initial density of 30,000 cells/well and cultured for 24 h. The invasive cells were counted in 10 random fields. The experiment was repeated at least three times and results are expressed relative to the number of control (100%). All the experiments were repeated three times. (* p < 0.05).

Article Snippet: The proteins were detected with the following antibodies: Rabbit anti-BKCa polyclone antibody (1:600, Alomone, Jerusalem, Israel), rabbit anti-caveolin-1 polyclone antibody (1:1000, Abcam).The primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (1:15000, Abcam), and the signals were developed using an enhanced chemiluminescence detection kit (Thermo).

Techniques: Expressing, Transfection, Cell Culture, Invasion Assay, Control