Journal: International journal of oncology

Article Title: Contribution of the PI3K/MMPs/Ln-5γ2 and EphA2/FAK/Paxillin signaling pathways to tumor growth and vasculogenic mimicry of gallbladder carcinomas.

doi: 10.3892/ijo.2013.1897

Figure Lengend Snippet: Figure 3. Expression of MMP-2, MT1-MMP proteins from sections [(A) SABC method, original magnification, x200] and supernates [(B) ELISA] of 3-D culture samples in vitro in GBC-SD, SGC-996 and GBC-SD+TIMP-2 groups. (A) The positive expression site of MMP-2 and MT1-MMP proteins presented yellow-brown reactant in the cytoplasm. Overexpression of MMP-2 (a1) and MT1-MMP (a4) proteins in GBC-SD group was observed. Expression of MMP-2 and MT1-MMP proteins in SGC-996 group (a2,5) and GBC-SD+TIMP-2 (a3,6) group was significantly decreased (*P<0.001, #P<0.01, vs. GBC-SD group). (B) Expression of MMP-2 (b1) and MT1-MMP (b2) proteins in GBC-SD group increased significantly with time, when compared with SGC-996 group and GBC-SD+TIMP-2 group (*P<0.001).

Article Snippet: The sections (4-μm) from each group were dehydrated in xylene and graded ethanol series, were added in order with primary antibody [MMP-2 (1: 200), MT1-MMP (1:100); rabbit polyclonal antibody, Wuhan Boster Co., China)], biotinylated secondary antibody, SABC reagents and DAB solution (Wuhan Boster Co.), respectively.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, In Vitro, Over Expression

Journal: International journal of oncology

Article Title: Contribution of the PI3K/MMPs/Ln-5γ2 and EphA2/FAK/Paxillin signaling pathways to tumor growth and vasculogenic mimicry of gallbladder carcinomas.

doi: 10.3892/ijo.2013.1897

Figure Lengend Snippet: Figure 4. Expression of MMP-2, MT1-MMP proteins/mRNAs from sections of tumor xenografts in vivo [(A) SABC method, original magnification, x200; (B) qRT-PCR)] in GBC-SD, SGC-996 and GBC-SD+TIMP-2 groups. Overexpression of MMP-2 (Aa1,7) and MT1-MMP (Aa4,7) proteins or mRNA (B) in GBC-SD group was observed in vivo. Expression of MMP-2 and MT1-MMP proteins or mRNA in SGC-996 group (Aa2,5,7 and B) and GBC-SD+TIMP-2 (Ac3,6,7 and B) group was significantly decreased (*P<0.001, vs. GBC-SD group).

Article Snippet: The sections (4-μm) from each group were dehydrated in xylene and graded ethanol series, were added in order with primary antibody [MMP-2 (1: 200), MT1-MMP (1:100); rabbit polyclonal antibody, Wuhan Boster Co., China)], biotinylated secondary antibody, SABC reagents and DAB solution (Wuhan Boster Co.), respectively.

Techniques: Expressing, In Vivo, Quantitative RT-PCR, Over Expression

Journal: Oncology Letters

Article Title: Co-localisation of intra-nuclear membrane type-1 matrix metalloproteinase and hypoxia inducible factor-2α in osteosarcoma and prostate carcinoma cells

doi: 10.3892/ol.2020.12419

Figure Lengend Snippet: Western blotting to measure MT1-MMP expression in total cell lysate and subcellular fractions. (A) MT1-MMP protein expression in total cell lysate in the fibrosarcoma HT1080 (positive control), osteosarcoma U2OS, breast carcinoma MDA-MB-231 and MCF-7 (negative control), and prostate carcinoma PC3 and LNCaP cell lines and human-derived BM-MSCs. The cancer cell lines in this panel express mainly the active form of MT1-MMP (57 kDa). (B) Subcellular fractionation of U2OS cells treated in 1% oxygen demonstrated increased nuclear expression of MT1-MMP compared with control. BM-MSCs, bone marrow mesenchymal stem cells; HIF, hypoxia-inducible factor; MT1-MMP, membrane type-1 matrix metalloproteinase.

Article Snippet: Slides were rinsed in running water and blocked in 4% BSA/PBS for 1 h. The MT1-MMP primary antibodies (mouse monoclonal anti-MMP-14 clone LEM-2/15.8, MAB3328 (Merck Millipore and rabbit polyclonal anti-HIF-2α (ab199) Abcam) were diluted to 1 in 50 in 4% BSA/PBS.

Techniques: Western Blot, Expressing, Positive Control, Negative Control, Derivative Assay, Fractionation, Control, Membrane

Journal: Oncology Letters

Article Title: Co-localisation of intra-nuclear membrane type-1 matrix metalloproteinase and hypoxia inducible factor-2α in osteosarcoma and prostate carcinoma cells

doi: 10.3892/ol.2020.12419

Figure Lengend Snippet: Confocal microscopy of U2OS cells following proximity ligation assays for MT1-MMP, HIF-1α and HIF-2α. (A) Demonstration of intra-nuclear presence of MT1-MMP. (B) There was strong signal for HIF-1α in 1% oxygen. (C) There was a slightly increased signal for HIF-2α in hypoxia. Scale bars, 50 µm. HIF, hypoxia-inducible factor; MT1-MMP, membrane type-1 matrix metalloproteinase.

Article Snippet: Slides were rinsed in running water and blocked in 4% BSA/PBS for 1 h. The MT1-MMP primary antibodies (mouse monoclonal anti-MMP-14 clone LEM-2/15.8, MAB3328 (Merck Millipore and rabbit polyclonal anti-HIF-2α (ab199) Abcam) were diluted to 1 in 50 in 4% BSA/PBS.

Techniques: Confocal Microscopy, Ligation, Membrane

Journal: Oncology Letters

Article Title: Co-localisation of intra-nuclear membrane type-1 matrix metalloproteinase and hypoxia inducible factor-2α in osteosarcoma and prostate carcinoma cells

doi: 10.3892/ol.2020.12419

Figure Lengend Snippet: Confocal microscopy following proximity ligation assays to assess protein-protein interactions between MT1-MMP and HIFs in U2OS and PC3 cells with BM-MSCs as a control. (A) There was some interaction between MT1-MMP and HIF-1α in 20% oxygen in both U2OS cells and MSCs. There was no significant interaction between MT1-MMP and HIF-1α in either cell type in 1% oxygen. There was evidence of intra-nuclear interaction between MT1-MMP and HIF-2α in U2OS and PC3 cells in 1% oxygen. Scale bars, 50 µm. Magnification, ×63. (B) Intra-nuclear interaction between MT1-MMP and HIF-2α in U2OS and prostate carcinoma PC3 cells treated with 1% oxygen was significant compared with control cells in 20% oxygen. *P=0.040 for U2OS and *P=0.028 for PC3 cells (Student's t-test). BM-MSCs, bone marrow mesenchymal stem cells; HIF, hypoxia-inducible factor; MT1-MMP, membrane type-1 matrix metalloproteinase.

Article Snippet: Slides were rinsed in running water and blocked in 4% BSA/PBS for 1 h. The MT1-MMP primary antibodies (mouse monoclonal anti-MMP-14 clone LEM-2/15.8, MAB3328 (Merck Millipore and rabbit polyclonal anti-HIF-2α (ab199) Abcam) were diluted to 1 in 50 in 4% BSA/PBS.

Techniques: Confocal Microscopy, Ligation, Protein-Protein interactions, Control, Membrane

Journal: Oncology Letters

Article Title: Co-localisation of intra-nuclear membrane type-1 matrix metalloproteinase and hypoxia inducible factor-2α in osteosarcoma and prostate carcinoma cells

doi: 10.3892/ol.2020.12419

Figure Lengend Snippet: Confirmation of intra-nuclear MT1-MMP in patient tumour cells using immunofluorescence and confocal microscopy with evidence of co-expression of MT1-MMP and HIF-2α. MT1-MMP (red dots) and HIF-2α (green dots) were present in the nucleus and cytoplasm of the sarcoma cells. There was evidence of co-localisation of MT1-MMP and HIF-2α in the nucleus (white arrows) and in the cytoplasm (white arrowheads). Scale bar, 10 µm. HIF, hypoxia-inducible factor; MT1-MMP, membrane type-1 matrix metalloproteinase.

Article Snippet: Slides were rinsed in running water and blocked in 4% BSA/PBS for 1 h. The MT1-MMP primary antibodies (mouse monoclonal anti-MMP-14 clone LEM-2/15.8, MAB3328 (Merck Millipore and rabbit polyclonal anti-HIF-2α (ab199) Abcam) were diluted to 1 in 50 in 4% BSA/PBS.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing, Membrane

Journal: Cells

Article Title: Role of Clathrin Light Chains in Regulating Invadopodia Formation

doi: 10.3390/cells10020451

Figure Lengend Snippet: Actin patches in CLCA−/− CLC−/− cells co-localize with common invadopodia markers. U373 AP2-GFP WT and CLTA−/− CLTB −/− cells were fixed 24 h post seeding. Cells were stained with an antibody against ( A ) Arp3 (red) ( B ) MMP14 ( D ) Cortactin (red) or transduced with ( C ) BacMam expressing mCherry-zyxin (red). Cells were analyzed by confocal microscopy for co-localization with AP2 (green) and actin (grey). Representative confocal images are shown. The scale bar represents 20 µm for the merge and 5 µm for the zoom region, respectively. Red arrows indicate co-localizing structures. ( E – H ) Pearson correlation analysis. Shown are the mean with SD. Statistical analysis: unpaired t -test. ARP3 8 fields of view, **** p < 0.0001; MMP14 13 fields of view, **** p < 0.0001; zyxin, 12 fields of view, ** p = 0.0014; Cortactin 11 fields of view, **** p < 0.0001.

Article Snippet: Rabbit polyclonal anti-MMP14 antibody (Abcam, ab51074) was used at 1:1000 for IF and WB and monoclonal antibody against β actin (A5441); used 1:5000 for WB).

Techniques: Staining, Transduction, Expressing, Confocal Microscopy

Journal: Cells

Article Title: Role of Clathrin Light Chains in Regulating Invadopodia Formation

doi: 10.3390/cells10020451

Figure Lengend Snippet: Knock out of CLCA and CLCB does not impair MMP14 internalization. ( A ) Schematic of microscopy based internalization assay. Cells plated on glass coverslips overnight were serum starved for 1 h, labelled with MMP14 antibody on ice for 15 min. For total surface MMP14 levels cells were directly fixed after primary antibody incubation and stained using the Alexa Fluor 647 labelled anti-rabbit secondary antibody (without permeabilization). For internal MMP14 levels cells were incubated at 37 °C for 30 min in order to start MMP14 internalization. For internal MMP14, cell surface was stripped using ice-cold PBS at pH 2.5, followed by fixation and immunostaining with permeabilization. Imaging was performed using confocal microscopy. Image sections were acquired every 0.5 µm for a total of 7.5 µm. Fiji was used to quantify MMP14 levels. ( B ) Representative confocal images for total surface and internal MMP14 fractions of U373 AP2-GFP. ( C ) Quantification of MMP14 signal intensities. Shown are the mean with SD. WT total surface 37 cells; WT internalized 38 cells; CLCA−/− CLCB−/− total surface 49 cells; CLCA−/− CLCB−/− internalized 51 cells. Shown are the mean with SD. Statistical analysis: unpaired t -test: total surface MMP14, **** p < 0.0001; internalized MMP14, **** p < 0.0001. ( D ) Intensity ratios from ( C ) of internalized MMP14/total surface MMP14 in U373 AP2-GFP WT and CLCA−/− CLCB−/− cells. Shown are the mean with SD. Statistical analysis: unpaired t -test: not significant (ns).

Article Snippet: Rabbit polyclonal anti-MMP14 antibody (Abcam, ab51074) was used at 1:1000 for IF and WB and monoclonal antibody against β actin (A5441); used 1:5000 for WB).

Techniques: Knock-Out, Microscopy, Incubation, Staining, Immunostaining, Imaging, Confocal Microscopy

Journal: Cells

Article Title: Role of Clathrin Light Chains in Regulating Invadopodia Formation

doi: 10.3390/cells10020451

Figure Lengend Snippet: MMP14 expression and internalization in U373 WT and CLCA−/−CLCB−/− cells. ( A ) Schematic of flow cytometry internalization assay. Cells were dissociated from cell culture flasks resuspended in serum free culture medium and kept on ice before incubation with saturating concentration of MMP14 antibody. Internalization was initiated by incubation at 37 °C for the respective time intervals between 0 and 40 min. After MMP14 internalization, cells were incubated with anti-rabbit Alexa Fluor 647 (1:10000) for 30min, washed and analyzed by flow cytometry (FACS Canto II, BD Biosciences). Data were analyzed using FlowJoTM version 10B). ( B ) Surface MMP14 was normalized to total MMP14. Relative percentage was calculated from 3 independent experiments. Median with SD is shown. Statistical analysis: unpaired t test,* p = 0.0111. ( C ) Flow cytometry-based MMP14 internalization assay. Shown are relative MMP14 surface levels normalized to 0 min. Each point represents mean and SD of three biological replicates.

Article Snippet: Rabbit polyclonal anti-MMP14 antibody (Abcam, ab51074) was used at 1:1000 for IF and WB and monoclonal antibody against β actin (A5441); used 1:5000 for WB).

Techniques: Expressing, Flow Cytometry, Cell Culture, Incubation, Concentration Assay

Journal: Cells

Article Title: Role of Clathrin Light Chains in Regulating Invadopodia Formation

doi: 10.3390/cells10020451

Figure Lengend Snippet: Fluorescence recovery after photobleaching of MMP14-pHluorin. ( A ) Representative live-cell confocal spinning disc microscopy of U373 WT and CLCA−/− CLCB−/− transiently expressing MMP14-pHluorin. Live-cell confocal imaging was performed for a total of 25 min at a frame rate of 5 s per frame. The scale bar equals 20 µm. Highlighted area indicates FRAP region. ( B ) Normalized fluorescence of FRAP recovery curves. Normalized mean fluorescence intensity profiles of WT (blue) and CLCA−/− CLCB−/− cells (red) before and after FRAP. Shown are mean and SD computed from 14 WT and 18 CLCA−/− CLCB−/− cells FRAP recovery curves. Standard deviations are shown. ( C ) t-Half of MMP14-pHluorin in U373 WT and CLCA−/− CLCB−/− cells. T-Half was calculated from 14 (WT) and 18 (CLCA−/− CLCB−/−) FRAP recovery events, each FRAP event was monitored in a different cell. Mean with SD are shown. Statistical analysis: unpaired t test, ns. All FRAP analysis was performed using the EasyFRAP-web tool. ( D ) Mobile fraction of MMP14-pHluorin in U373 WT and CLCA−/− CLCB−/− cells. Mobile fraction was calculated from 14 (WT) and 18 (CLCA−/− CLCB−/−) FRAP recovery events. Mean with SD are shown. Statistical analysis: unpaired t test, ** p = 0.0069. ( D ) t-Half of MMP14-pHluorin in U373 WT and CLCA−/− CLCB−/− cells. T-Half was calculated from 14 (WT) and 18 (CLCA−/− CLCB−/−) FRAP recovery events, each FRAP event was monitored in a different cell. Mean with SD are shown. Statistical analysis: unpaired t test, not significant (ns). All FRAP analysis was performed using the EasyFRAP-web tool.

Article Snippet: Rabbit polyclonal anti-MMP14 antibody (Abcam, ab51074) was used at 1:1000 for IF and WB and monoclonal antibody against β actin (A5441); used 1:5000 for WB).

Techniques: Fluorescence, Microscopy, Expressing, Imaging

Journal: Cells

Article Title: Role of Clathrin Light Chains in Regulating Invadopodia Formation

doi: 10.3390/cells10020451

Figure Lengend Snippet: MMP14 attenuation in CLCA−/− CLCB−/− cells prevents invadopodia formation. ( A ) Western blot analysis of WT and CLCA−/− CLCB−/− cell lines showing the protein levels of cells stably expressing Mock, scrambled shRNA or an MMP14 shRNA construct. Actin protein levels were used as control. ( B ) Representative widefield microscopy images of WT and CLCA−/− CLCB−/− cells stably expressing scrambled shRNA or an MMP14 shRNA construct, scale bar indicates 20 µm. ( C ) Quantification of relative percentage of actin phenotype among U373 WT and CLCA−/− CLCB−/− cells stably expressing scrambled shRNA or an MMP14 shRNA construct. 10 fields of view were analyzed. WT shscrmbld 23 cells, WT shMMP14 13 cells, CLCA−/− CLCB−/− shscrmbld 25 cells, CLCA−/− CLCB−/− shMMP14 21 cells.

Article Snippet: Rabbit polyclonal anti-MMP14 antibody (Abcam, ab51074) was used at 1:1000 for IF and WB and monoclonal antibody against β actin (A5441); used 1:5000 for WB).

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Construct, Control, Microscopy

Journal: Cells

Article Title: Role of Clathrin Light Chains in Regulating Invadopodia Formation

doi: 10.3390/cells10020451

Figure Lengend Snippet: Loss of CLCs induces invadopodia formation by actin polymerization and MMP14 recruitment. Schematic showing upregulation of MMP14 recycling in CLCA−/− CLCB−/− cells causing invadopodia formation.

Article Snippet: Rabbit polyclonal anti-MMP14 antibody (Abcam, ab51074) was used at 1:1000 for IF and WB and monoclonal antibody against β actin (A5441); used 1:5000 for WB).

Techniques: