human caix protein  (Sino Biological)

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: Identification of the CAIX Interactome using the BioID proximal ligation strategy and validation of high confidence proximal CAIX-associating proteins by co-immunoprecipitation. ( a ) Schematic showing the domain structure of the CAIX-BirA* fusion protein used for the BioID studies. SP, signal peptide; PG, proteoglycan-like domain; CA, catalytic domain; TM, transmembrane domain; IC, intracellular domain. ( b ) FACS analysis showing levels of constitutive expression of the CAIX-BirA* fusion protein expressed by MDA-MB-231 cells (MDA-MB-231-CAIX-BirA*) cultured in normoxia compared to levels of endogenous CAIX expression by WT MDA-MB-231 cells cultured in either normoxia or hypoxia. The filled (gray) histograms indicate specific CAIX staining whereas the dotted lined histograms indicate staining by isotype-specific control IgG. ( c ) Image showing exogenous CAIX (red) localized at the cell surface of MDA-MB-231-CAIX-BirA* cells grown in normoxia. Scale bar, 10 μ M . ( d – g ) Co-IP of high confidence proximal CAIX-associating proteins ( d ) ITGB1, ( e ) ITGA2 ( f ) CD98hc and ( g ) MMP14 with exogenous CAIX expressed by MDA-MB-231-CAIX-BirA* cells cultured in normoxia. ( h – j ) Co-IP of ( h ) ITGB1, ( i ) ITGA2 and ( j ) MMP14 with endogenous CAIX expressed by WT MDA-MB-231 cells cultured in either normoxia (N) or hypoxia (H). Isotype-specific IgG was used as a control for co-IPs and data in ( d ) to ( j ) are representative of at least two independent experiments. IgG, immunoglobulin G.

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Ligation, Biomarker Discovery, Immunoprecipitation, Expressing, Cell Culture, Staining, Control, Co-Immunoprecipitation Assay

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: Selected CAIX-BirA*FLAG high-confidence proximal associating proteins identified by BioID in MDA-MB-231 cells

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques:

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX colocalizes with integrins and MMP14 in pseudopodia-like protrusions resembling lamellipodia. ( a ) Images of MDA-MB-231-CAIX-BirA* cells cultured on type 1 collagen in normoxia, showing colocalization (yellow, arrows in merge) of exogenously expressed CAIX (red) with ITGB1, ITGA2, MMP14 and cofilin (each marker in green), but not with FAK (green; arrowheads). Scale bar, 10 μm. ( b ) Images of WT MDA-MB-231 cells grown in either normoxia or hypoxia, showing colocalization of hypoxia-induced CAIX (red) with F-actin (green) in pseudopodia-like protrusions (arrows, merge). Scale bar, 10 μm. ( c ) Images of WT MDA-MB-231 cells cultured on type 1 collagen in hypoxia, showing colocalization of hypoxia-induced CAIX (red) with F-actin, ITGA2 and MMP14 (green) at leading edges of cells (arrows, merge), but not at focal adhesions. Scale bar, 10 μm. ( d and e ) 3D maximum projections of 2D images of MDA-MB-231-CAIX-BirA* cells transfected with GFP and cultured on type 1 collagen in normoxia, showing robust colocalization of CAIX (red) with ( d ) ITGA2 and ( e ) MMP14 (blue) in pseudopodia-like protrusions and depletion of cytoplasmic GFP (green) from these regions. Images for each channel are shown in the upper panels and merged images are shown in the lower panels. Scale bar, 10 μm.

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Cell Culture, Marker, Transfection

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX regulates migration of breast cancer cells. ( a ) Images of wound-induced cell migration by the indicated MDA-MB-231 cells cultured in normoxia. Scale bar, 300 μm. Dashed lines demarcate the wound boundary at t =0 h and yellow shading indicates the cell-free area. ( b ) Quantification of migration by the cells described in a . *** P <0.001. ( c ) Images of wound-induced migration of MDA-MB-231 CAIX-BirA* cells cultured in normoxia and treated with increasing concentrations of U-104. Scale bar, 300 μm. Wound boundaries and cell-free area are demarcated as described in a . ( d ) Quantification of migration by the cells described in c . * P <0.05, *** P <0.001. Data in panels ( b ) and ( d ) show the mean±s.e.m. of technical replicates ( n =16) and are representative of two independent experiments. ( e ) Wound-induced migration of WT MDA-MB-231 cells cultured in normoxia (black bars) or hypoxia (gray bars) and treated with U-104. Data show the mean±s.e.m. of technical replicates ( n =12) and are representative of three independent experiments. ** P <0.01, *** P <0.001. Statistical analysis was performed using Student’s t -test ( b ) or ANOVA ( d , e ).

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Migration, Cell Culture

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX colocalizes with MMP14 at invadopodia and regulates type I collagen degradation. ( a ) Images of a MDA-MB-231-CAIX-BirA* cell (white dashed line shows boundary) cultured on type I collagen in normoxia, identifying a ROI (yellow box) containing invadopodia (arrows) labeled for Cortactin (cyan), CAIX (red) and MMP14 (green). Profile plots showing colocalization of CAIX and MMP14 at cortactin-positive invadopodia (1 and 2) along the indicated lines (insets) are shown at the right. Scale bar, 10 μm. ( b ) Images of an MDA-MB-231-CAIX-BirA* cell (white dashed line shows cell boundary) cultured on DQ type I collagen in normoxia, identifying an ROI (yellow box) containing regions of collagen degradation (1 and 2, arrows) that are labeled for DQ collagen (cyan), CAIX (red) and MMP14 (green). Profile plots showing colocalization of the signals along the indicated lines (insets) are shown to the right. Scale bar, 10 μm. ( c ) Images showing WT MDA-MB-231 cells cultured in hypoxia on fluorescently labeled gelatin (blue, with black areas denoting regions of degradation) and stained for CAIX (green) and markers of invadopodia (red), including cortactin, Tks5 and MMP14 to demonstrate colocalization at mature invadopodia (white arrows; yellow foci). Scale bars, 10 μm (left), 2 μm (right). ( d ) Quantification of DQ collagen degradation by the indicated MDA-MB-231 cell lines grown in normoxia. *** P <0.001. ( e ) Quantitation of degradation of DQ collagen by MDA-MB-231 CAIX KO and CAIX rescue cells cultured in normoxia. * P <0.05. Data in ( d ) and ( e ) show the mean±s.e.m. of technical replicates ( n =5) and are representative of three independent experiments. ( f ) Dose-dependent inhibition of DQ collagen degradation by CAIX rescue cells treated with U-104. Data show the mean±s.e.m. of technical replicates ( n =4) and are representative of two independent experiments. * P <0.05, ** P <0.01. ( g ) Quantification of DQ collagen degradation by WT MDA-MB-231 and CAIX KO cells cultured in hypoxia. Data show the mean±s.e.m. of technical replicates ( n =8) and are representative of three independent experiments. ** P <0.01. ( h and i ) Invasion through Matrigel by highly metastatic MDA-MB-231 LM2-4 breast cancer cells cultured in hypoxia and ( h ) expressing CAIX-targeted (shCAIX) shRNA or ( i ) treated with U-104. Data show the mean±s.e.m. of three independent experiments. *** P <0.001. Western blots of hypoxia-induced CAIX expression are shown as insets.

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Cell Culture, Labeling, Staining, Quantitation Assay, Inhibition, Expressing, shRNA, Western Blot

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX regulates breast cancer cell invasion and metastasis. ( a ) Schematic showing the domain structure of wild type (WT) huCAIX (459 aa) and constructs lacking either the intracellular domain (ΔIC), the extracellular proteoglycan-like domain (ΔPG) or the indicated point mutations in the IC domain. SP, signal peptide; CA, catalytic domain; TM, transmembrane domain. ( b ) Western blot analysis of the levels of expression of the indicated huCAIX constructs by 4T1shCAIX cells cultured in hypoxia. β-actin served as a loading control. ( c ) Analysis of CAIX catalytic activity using the in-cell carbonic anhydrase activity assay. Assays were performed in normoxia in the presence or absence of U-104 (50 μ M ) as indicated. Levels of CAIX catalytic activity were normalized by using the time required to achieve 50% of the total decrease in pH. Data were normalized to the spontaneous rate of reaction in the presence of buffer alone and the activity of cells expressing WT huCAIX was set to 100%. Data show the mean±s.e.m. of technical replicates ( n =3/group) and are representative of three independent experiments * P <0.05, *** P <0.001. ( d ) Invasion through Matrigel by the indicated 4T1 cell lines cultured in hypoxia. Data show the mean±s.e.m. of three independent experiments. * P <0.05, *** P <0.001. ( e ) Invasion through Matrigel by the 4T1shCAIX cell lines expressing WT, ΔIC and ΔPG variants of huCAIX and cultured in hypoxia. * P <0.05, *** P <0.001. ( f ) Invasion through Matrigel by 4T1 sh/WT huCAIX cells cultured in hypoxia and treated with U-104. *** P <0.001. Western blots of hypoxia-induced CAIX expression are shown as insets. For ( d )– ( f ), data show the mean±s.e.m. of three independent experiments. ( g – i ) Analysis of invasion through type 1 collagen by 4T1 sh/WT huCAIX cells cultured in hypoxia and treated with ( g ) U-104 (50 μ M ), ( h ) anti-MMP14 antibody (20 μg/ml) or ( i ) a combination of anti-MMP14 antibody (20 μg/ml) and U-104 (50 μ M ). * P <0.05, ** P <0.01. Data in ( g )–( i ) show the mean±s.e.m. of three independent experiments. ( j ) Analysis of spontaneous lung metastases formed by the indicated 4T1 cell lines following growth of orthotopic breast tumors. Data show the mean±s.e.m. n =6/group. ( k ) Analysis of experimental lung metastases formed by the indicated 4T1 cell lines. Mean±s.e.m. is shown. n =6/group. * P <0.05, ** P <0.01. Statistical analysis was performed using ANOVA ( c , d , e , k ) or Student’s t -test ( f , g , h ).

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Construct, Western Blot, Expressing, Cell Culture, Control, Activity Assay

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX associates with MMP14 through its intracellular domain and potentiates MMP14 activity. ( a ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT, ΔIC or ΔPG variants of huCAIX. ( b ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT huCAIX or the T443A point mutant. ( c ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT or the Y449A point mutant. ( d ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells grown in hypoxia and expressing WT huCAIX, the S448A point mutant or the ΔPG mutant. ( e ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells in hypoxia expressing WT huCAIX, the ΔIC truncation or the double point mutant S448A+Y449A. Isotype-specific IgG was used as a control for co-IPs. ( f ) Time-dependent stimulation of MMP14 activity in the presence (red bars) or absence (black bars) of equimolar concentrations of CAIX. Data show the mean±s.e.m. of technical replicates ( n =5) and are representative of two independent experiments. * P <0.05, ** P <0.01. ( g ) Dose-dependent inhibition of CAIX-mediated stimulation of MMP14 activity in the presence of increasing concentrations of U-104. Mean±s.e.m. of technical replicates ( n =5). Data are representative of two independent experiments. *** P <0.001. ( h ) Inhibition of CAIX-mediated stimulation of MMP14 activity in the presence of increasing amounts of D 2 O (heavy water). Mean±s.e.m. of technical replicates ( n =5). Data are representative of three independent experiments. *** P <0.001. Statistical analysis was performed using Student’s t -test ( f ) or ANOVA ( g , h ).

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Cell Culture, Expressing, Mutagenesis, Control, Inhibition

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX stimulates the activity of MMP14. Model for CAIX-mediated regulation of MMP14 activity. CAIX, which is upregulated in hypoxia, associates through its intracellular domain with MMP14 at functional invadopodia. At these invasive structures, CAIX functions to potentiate MMP14-mediated collagen degradation by directly contributing H + ions required for MMP14 catalytic activity.

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Activity Assay, Functional Assay