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MMP-9 downregulation is critical in IB-induced metastasis inhibition in <t>human</t> <t>TNBC</t> cells. ( a ) After treatment with 1 or 3 µM IB, wound healing scratch assays were performed using TNBC cells. Cell migration was monitored under a phase-contrast microscope for 24 h: Bar, 50 μm. Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. 24 h untreated control. ( b ) Invasion assays of TNBC cells treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are presented as the mean ± SD. *** p < 0.001 vs. untreated control. ( c ) Cell extracts were prepared from cells treated with the indicated IB concentrations for 24 h. MMP-9 and MMP-2 levels were detected using Western blotting. α-tubulin was used as a loading control. ( d ) TNBC cells were treated with the indicated IB concentrations for 24 h. Cell extracts were prepared for Western blotting of mesenchymal markers. ( e ) MMP-9 overexpressing stable cells were scratched during wound healing assays. MMP-9 upregulation was confirmed using Western blotting. Cell migration was monitored for 24 h and observed under a phase-contrast microscope: Bar, 50 μm. The graphs quantitatively show the area of wound recovery. Data are presented as the mean ± SD. *** p < 0.0001 vs. IB-treated vector cells. ( f ) Invasion assays of MMP-9 overexpressing stable cell lines treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. IB-treated vector cells.
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MMP-9 downregulation is critical in IB-induced metastasis inhibition in <t>human</t> <t>TNBC</t> cells. ( a ) After treatment with 1 or 3 µM IB, wound healing scratch assays were performed using TNBC cells. Cell migration was monitored under a phase-contrast microscope for 24 h: Bar, 50 μm. Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. 24 h untreated control. ( b ) Invasion assays of TNBC cells treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are presented as the mean ± SD. *** p < 0.001 vs. untreated control. ( c ) Cell extracts were prepared from cells treated with the indicated IB concentrations for 24 h. MMP-9 and MMP-2 levels were detected using Western blotting. α-tubulin was used as a loading control. ( d ) TNBC cells were treated with the indicated IB concentrations for 24 h. Cell extracts were prepared for Western blotting of mesenchymal markers. ( e ) MMP-9 overexpressing stable cells were scratched during wound healing assays. MMP-9 upregulation was confirmed using Western blotting. Cell migration was monitored for 24 h and observed under a phase-contrast microscope: Bar, 50 μm. The graphs quantitatively show the area of wound recovery. Data are presented as the mean ± SD. *** p < 0.0001 vs. IB-treated vector cells. ( f ) Invasion assays of MMP-9 overexpressing stable cell lines treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. IB-treated vector cells.
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MMP-9 downregulation is critical in IB-induced metastasis inhibition in <t>human</t> <t>TNBC</t> cells. ( a ) After treatment with 1 or 3 µM IB, wound healing scratch assays were performed using TNBC cells. Cell migration was monitored under a phase-contrast microscope for 24 h: Bar, 50 μm. Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. 24 h untreated control. ( b ) Invasion assays of TNBC cells treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are presented as the mean ± SD. *** p < 0.001 vs. untreated control. ( c ) Cell extracts were prepared from cells treated with the indicated IB concentrations for 24 h. MMP-9 and MMP-2 levels were detected using Western blotting. α-tubulin was used as a loading control. ( d ) TNBC cells were treated with the indicated IB concentrations for 24 h. Cell extracts were prepared for Western blotting of mesenchymal markers. ( e ) MMP-9 overexpressing stable cells were scratched during wound healing assays. MMP-9 upregulation was confirmed using Western blotting. Cell migration was monitored for 24 h and observed under a phase-contrast microscope: Bar, 50 μm. The graphs quantitatively show the area of wound recovery. Data are presented as the mean ± SD. *** p < 0.0001 vs. IB-treated vector cells. ( f ) Invasion assays of MMP-9 overexpressing stable cell lines treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. IB-treated vector cells.
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MMP-9 downregulation is critical in IB-induced metastasis inhibition in <t>human</t> <t>TNBC</t> cells. ( a ) After treatment with 1 or 3 µM IB, wound healing scratch assays were performed using TNBC cells. Cell migration was monitored under a phase-contrast microscope for 24 h: Bar, 50 μm. Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. 24 h untreated control. ( b ) Invasion assays of TNBC cells treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are presented as the mean ± SD. *** p < 0.001 vs. untreated control. ( c ) Cell extracts were prepared from cells treated with the indicated IB concentrations for 24 h. MMP-9 and MMP-2 levels were detected using Western blotting. α-tubulin was used as a loading control. ( d ) TNBC cells were treated with the indicated IB concentrations for 24 h. Cell extracts were prepared for Western blotting of mesenchymal markers. ( e ) MMP-9 overexpressing stable cells were scratched during wound healing assays. MMP-9 upregulation was confirmed using Western blotting. Cell migration was monitored for 24 h and observed under a phase-contrast microscope: Bar, 50 μm. The graphs quantitatively show the area of wound recovery. Data are presented as the mean ± SD. *** p < 0.0001 vs. IB-treated vector cells. ( f ) Invasion assays of MMP-9 overexpressing stable cell lines treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. IB-treated vector cells.
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MMP-9 downregulation is critical in IB-induced metastasis inhibition in human TNBC cells. ( a ) After treatment with 1 or 3 µM IB, wound healing scratch assays were performed using TNBC cells. Cell migration was monitored under a phase-contrast microscope for 24 h: Bar, 50 μm. Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. 24 h untreated control. ( b ) Invasion assays of TNBC cells treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are presented as the mean ± SD. *** p < 0.001 vs. untreated control. ( c ) Cell extracts were prepared from cells treated with the indicated IB concentrations for 24 h. MMP-9 and MMP-2 levels were detected using Western blotting. α-tubulin was used as a loading control. ( d ) TNBC cells were treated with the indicated IB concentrations for 24 h. Cell extracts were prepared for Western blotting of mesenchymal markers. ( e ) MMP-9 overexpressing stable cells were scratched during wound healing assays. MMP-9 upregulation was confirmed using Western blotting. Cell migration was monitored for 24 h and observed under a phase-contrast microscope: Bar, 50 μm. The graphs quantitatively show the area of wound recovery. Data are presented as the mean ± SD. *** p < 0.0001 vs. IB-treated vector cells. ( f ) Invasion assays of MMP-9 overexpressing stable cell lines treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. IB-treated vector cells.

Journal: International Journal of Molecular Sciences

Article Title: Ibulocydine Inhibits Migration and Invasion of TNBC Cells via MMP-9 Regulation

doi: 10.3390/ijms25116123

Figure Lengend Snippet: MMP-9 downregulation is critical in IB-induced metastasis inhibition in human TNBC cells. ( a ) After treatment with 1 or 3 µM IB, wound healing scratch assays were performed using TNBC cells. Cell migration was monitored under a phase-contrast microscope for 24 h: Bar, 50 μm. Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. 24 h untreated control. ( b ) Invasion assays of TNBC cells treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are presented as the mean ± SD. *** p < 0.001 vs. untreated control. ( c ) Cell extracts were prepared from cells treated with the indicated IB concentrations for 24 h. MMP-9 and MMP-2 levels were detected using Western blotting. α-tubulin was used as a loading control. ( d ) TNBC cells were treated with the indicated IB concentrations for 24 h. Cell extracts were prepared for Western blotting of mesenchymal markers. ( e ) MMP-9 overexpressing stable cells were scratched during wound healing assays. MMP-9 upregulation was confirmed using Western blotting. Cell migration was monitored for 24 h and observed under a phase-contrast microscope: Bar, 50 μm. The graphs quantitatively show the area of wound recovery. Data are presented as the mean ± SD. *** p < 0.0001 vs. IB-treated vector cells. ( f ) Invasion assays of MMP-9 overexpressing stable cell lines treated with 0.5 or 1 µM IB for 24 h were performed. Invading cells were stained with crystal violet and observed using a fluorescence microscope (4×). Data are shown as the mean ± SD. * p < 0.05, *** p < 0.001 vs. IB-treated vector cells.

Article Snippet: The human TNBC cell lines (Hs578T and MDA-MB-435S) were purchased from the American Tissue Culture Collection (ATCC, VA, USA).

Techniques: Inhibition, Migration, Microscopy, Control, Staining, Fluorescence, Western Blot, Plasmid Preparation, Stable Transfection