Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: S chematic representation of the workflow used in this study to generate anti-CAIX mAbs. a) Cartoon of the rhCAIX ECD sequence (NP_001207.2) used: SP , signal peptide (gray); PG , proteoglycan-like domain (red); CA , catalytic domain (blue); His , penta-histidine tag (green) used for IMAC purification. b) Workflow of mAb production, characterization and selection process; for details see text

Article Snippet: Primary antibodies included anti-human CAIX (M75, Bioscience; 1:200), anti-human CAIX (MAB2188, R&D Systems, 1:200), anti-cleaved caspase 3 (Asp175-5A1E, Cell Signaling, 1:100), and novel anti-CAIX antibodies reported herein, including 11H9, c11H9, 12H8, c12H8, 2C7, c2C7, c2D7, 4A2, m4A2, 9B6 and m9B6 (all diluted to 10 μg/mL).

Techniques: Sequencing, Purification, Selection

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Binding of selected recombinant anti-CAIX antibodies by Surface Plasmon Resonance. Six (6) hybridoma derived mAbs were selected through screening (see , Suppl. Fig. S2A-C), sequenced, recloned in the appropriate IgG framework and recombinantly expressed in CHO cells (for details see text). (a) Purified recombinant antibodies (c11H9, c12H8, c2C7, m4A2, m9B6, c2D7) were captured with the appropriate anti-Fc surface (anti-human Fc: c11H9, c12H8, c2C7, c2D7; anti-mouse Fc: m4A2, m9B6). cG250 was used as a benchmark. Serial dilutions (0.74–60 nM) of rhCAIX monomer (CAIX-M) and dimer (CAIX-D) were then injected, followed by a buffer injection. Sensorgrams were aligned, double-referenced, and fitted to the 1:1 binding model to calculate ka, kd, KD and RUmax when flowing CAIX-M and apparent ka, kd, KD and RUmax when flowing CAIX-D (see ). (b) Graphs depicting changes in the calculated ka, kd, KD and RUmax when flowing rhCAIX-M versus rhCAIX-D over the immobilized antibodies

Article Snippet: Primary antibodies included anti-human CAIX (M75, Bioscience; 1:200), anti-human CAIX (MAB2188, R&D Systems, 1:200), anti-cleaved caspase 3 (Asp175-5A1E, Cell Signaling, 1:100), and novel anti-CAIX antibodies reported herein, including 11H9, c11H9, 12H8, c12H8, 2C7, c2C7, c2D7, 4A2, m4A2, 9B6 and m9B6 (all diluted to 10 μg/mL).

Techniques: Binding Assay, Recombinant, SPR Assay, Derivative Assay, Purification, Injection

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Representative kinetic and apparent kinetic values for binding of recombinant anti-CAIX antibodies obtained by SPR analysis and by flowing rhCAIX monomer or dimer, respectively. Antibodies suitable for ADC, CAIX enzyme inhibitor and imaging/detection are listed in green, blue and red, respectively. The cG250 mAb was used as control (black)

Article Snippet: Primary antibodies included anti-human CAIX (M75, Bioscience; 1:200), anti-human CAIX (MAB2188, R&D Systems, 1:200), anti-cleaved caspase 3 (Asp175-5A1E, Cell Signaling, 1:100), and novel anti-CAIX antibodies reported herein, including 11H9, c11H9, 12H8, c12H8, 2C7, c2C7, c2D7, 4A2, m4A2, 9B6 and m9B6 (all diluted to 10 μg/mL).

Techniques: Binding Assay, Recombinant, Imaging, Control

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Evaluation of anti-hCAIX antibodies for inhibition of catalytic activity of CAIX. (a) In vitro assay assessing rhCAIX enzyme activity (rhCAIX dimer: 0.5 µM) as a function of 4-MUA (100 µM) substrate-released fluorescence over time in the presence of m4A2 (1 µM, blue open square) or m9B6 (1 µM, blue open circle). Murine IgG CTL mAb (1 µM, red open square) and SMI Acetazolamide (10 µM, black square) were used as negative and positive controls, respectively. (b) Kinetic analysis of the activity of rhCAIX dimer using the physiologic substrate of CAIX, CO 2 , in response to increasing concentrations of m4A2. Normal mouse IgG and non-function-blocking antibody c2D7 were used as negative controls. The spontaneous change in pH in absence of rhCAIX dimer is shown for reference. Data are presented as the mean ± s.e.m. of technical replicates (n = 3/group) and are representative of 2 independent experiments. (c) Quantification of the relative percent activity of rhCAIX dimer in response to increasing concentrations of m4A2, calculated from the AUC of data in panel B . Data are presented as the mean ± s.e.m. of technical replicates (n = 3/group) and are representative of 2 independent experiments (*** P < .001; ANOVA)

Article Snippet: Primary antibodies included anti-human CAIX (M75, Bioscience; 1:200), anti-human CAIX (MAB2188, R&D Systems, 1:200), anti-cleaved caspase 3 (Asp175-5A1E, Cell Signaling, 1:100), and novel anti-CAIX antibodies reported herein, including 11H9, c11H9, 12H8, c12H8, 2C7, c2C7, c2D7, 4A2, m4A2, 9B6 and m9B6 (all diluted to 10 μg/mL).

Techniques: Inhibition, Activity Assay, In Vitro, Fluorescence, Blocking Assay

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Assessment of recombinant anti-hCAIX antibodies as tools for immunohistochemistry and in vivo imaging applications. (a) Immunohistochemical staining for expression of CAIX in FFPE tissue sections from PK-8 human PDAC xenografts using recombinant antibodies c11H9, m9B6 and c2D7. Commercial anti-CAIX mAb, M75, was used as benchmark-based positive control. Scale bar, 100 μm; inset, 20 μm. Anti-CAIX antibody 11H9 (b) and a (c) control antibody (IgG) were conjugated to the chelator pSCN-Bn-DTPA and radiolabelled with 111 In. Conjugates were administered to NODSCID IL2RKO mice bearing subcutaneous hCAIX-positive HT-29 colorectal cancer xenografts (100 mm 3 ). Uptake, accumulation and retention of the radiolabelled 11H9 and IgG control were monitored 24–168 h post-injection by SPECT/CT imaging (B, C; see also ) (t, tumor; l, lung; s, stomach; h, heart)

Article Snippet: Primary antibodies included anti-human CAIX (M75, Bioscience; 1:200), anti-human CAIX (MAB2188, R&D Systems, 1:200), anti-cleaved caspase 3 (Asp175-5A1E, Cell Signaling, 1:100), and novel anti-CAIX antibodies reported herein, including 11H9, c11H9, 12H8, c12H8, 2C7, c2C7, c2D7, 4A2, m4A2, 9B6 and m9B6 (all diluted to 10 μg/mL).

Techniques: Recombinant, Immunohistochemistry, In Vivo Imaging, Immunohistochemical staining, Staining, Expressing, Positive Control, Control, Injection, Single Photon Emission Computed Tomography, Imaging

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Evaluation of internalization and ADC potential of the CAIX antibodies in vitro (a) Representative processed images of SK-RC-52 cells incubated with serial dilutions of pHAb-labeled c2C7 (bottom) and hIgG-pHAb control (middle). Untreated cells (top) were used as background control (yellow, antibody; blue, nuclei). (b) Internalization dose-response (0–20 µg/mL) of pHAb-labeled mAbs by SK-RC-52 cells that were ‘coated’ with mAbs for 1h at 4°C, washed and then transferred to 37°C for 24 h. Cells were imaged using the ImageXpress Micro (IXM) Widefield High Content Screening System, and images were processed using MetaXpress imaging software and further analyzed using Graphpad Prism v8. (c) Calculation of the total amount of internalized pHAb-labeled mAb (10 μg/mL, at t = 24 h) by SK-RC-52 cells, corrected for nonspecific internalization and expressed as fold change from t = 0 h. For all graphs, ADC candidates (c11H9, c12H8, c2C7), enzyme inhibitors (m4A2, m9B6), the imaging/detection antibody (c2D7), and the control antibody are depicted in green, blue, red, and black respectively

Article Snippet: Primary antibodies included anti-human CAIX (M75, Bioscience; 1:200), anti-human CAIX (MAB2188, R&D Systems, 1:200), anti-cleaved caspase 3 (Asp175-5A1E, Cell Signaling, 1:100), and novel anti-CAIX antibodies reported herein, including 11H9, c11H9, 12H8, c12H8, 2C7, c2C7, c2D7, 4A2, m4A2, 9B6 and m9B6 (all diluted to 10 μg/mL).

Techniques: In Vitro, Incubation, Labeling, Control, High Content Screening, Imaging, Software

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Evaluation of the ADC potential of the CAIX antibodies in vitro . (a) Analysis of expression of hCAIX by 67NR mouse breast cancer cells engineered to constitutively express hCAIX (CAIX-pos), compared to parental, hCAIX-negative (CAIX-neg) cells. Top panel, immunoblot for CAIX. Vinculin was used as a loading control. Bottom panel, immunofluorescence images. Scale bar, 10 μm. (b) Viability of cells described in panel A cultured with increasing concentrations of DM1-conjugated (blue graphs) and non-conjugated (black graphs) CAIX mAbs or a nonspecific IgG CTL antibody. Data show the mean ± s.e.m. of technical replicates (n = 3/group) and are representative of 2 independent experiments. Representative IC50 values were calculated using Graphpad Prism v8. (c) Analysis of expression of hCAIX by MIA PaCa-2 human PDAC cells engineered to constitutively express hCAIX (CAIX-pos), compared to hCAIX-negative (CAIX-neg) control cells. Top panel, immunoblot for CAIX. Vinculin was used as a loading control. Bottom panel, immunofluorescence images. Scale bar, 20 μm. (d) Viability of cells described in panel C cultured with increasing concentrations of c2C7-DM1 (left) or non-conjugated c2C7 mAb (right) (blue graphs, CAIX pos cells; black graphs, CAIX negative cells). Data show the mean ± s.e.m. of technical replicates (n = 3/group) and are representative of 2 independent experiments. Representative IC50 values were calculated using Graphpad Prism v8

Article Snippet: Primary antibodies included anti-human CAIX (M75, Bioscience; 1:200), anti-human CAIX (MAB2188, R&D Systems, 1:200), anti-cleaved caspase 3 (Asp175-5A1E, Cell Signaling, 1:100), and novel anti-CAIX antibodies reported herein, including 11H9, c11H9, 12H8, c12H8, 2C7, c2C7, c2D7, 4A2, m4A2, 9B6 and m9B6 (all diluted to 10 μg/mL).

Techniques: In Vitro, Expressing, Western Blot, Control, Immunofluorescence, Cell Culture

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Administration of c2C7-DM1 ADC to mice bearing CAIX-positive MIA PaCa-2 tumors increases tumor cell death. (a) Representative images of tumor tissue sections from CAIX-positive MIA PaCa-2 PDAC xenografts stained for expression of CAIX. Scale bar, 100 μm; inset, 20 μm. (b-d) Mice bearing subcutaneous CAIX-positive MIA PaCa-2 PDAC xenografts were administered increasing doses of c2C7-DM1 (n = 8 mice/group). (b) Study timeline and tumor growth curve. *** P < .001; ** P < .01; two-way ANOVA. (c) Tumor growth when control mice harboring tumors and administered vehicle reached the study endpoint. *** P < .001; * P < .05; two-way ANOVA. (d) Survival analysis of tumor-bearing mice administered 10 mg/kg c2C7-DM1, compared to mice given vehicle control. (e) Representative images of tumor tissue sections from CAIX-positive MIA PaCa-2 xenografts administered increasing doses of c2C7-DM1 and stained for cleaved caspase 3, a marker of apoptosis (black arrows). Scale bar, 50 μm; inset, 10 μm. (f) Quantification of cleaved caspase 3 (n = 5, each 5 fields). *** P < .001; * P < .05; two-way ANOVA. (g) Representative images of tumor tissue sections from CAIX-positive MIA PaCa-2 xenografts described in panel A stained with H&E to evaluate regions of necrosis. Boundaries between viable and necrotic tissue () are denoted with a black line. Scale bar, 100 μm. (h) Quantification of necrosis (n = 4, each 5 fields). *** P < .001; * P < .05; two-way ANOVA

Article Snippet: Primary antibodies included anti-human CAIX (M75, Bioscience; 1:200), anti-human CAIX (MAB2188, R&D Systems, 1:200), anti-cleaved caspase 3 (Asp175-5A1E, Cell Signaling, 1:100), and novel anti-CAIX antibodies reported herein, including 11H9, c11H9, 12H8, c12H8, 2C7, c2C7, c2D7, 4A2, m4A2, 9B6 and m9B6 (all diluted to 10 μg/mL).

Techniques: Staining, Expressing, Control, Marker

Journal: Cancers

Article Title: Attenuation of the Hypoxia Inducible Factor Pathway after Oncolytic Adenovirus Infection Coincides with Decreased Vessel Perfusion

doi: 10.3390/cancers12040851

Figure Lengend Snippet: Infection foci proximal to hypoxic areas are associated with low vascular endothelial growth factor ( VEGF ) expression while adenovirus infection down-regulates HIF-1α in vitro. Hypoxic areas in DLD-1 xenograft were labelled with pimonidazole 16 days after intravenous administration of EnAd. ( A – C ) Serial sections were stained for Hexon (brown) and either pimonidazole adducts (I, red) or VEGF RNA (II, red puncta). A non-infected area ( A ), an area containing a virus distant to hypoxia ( B ) and two virus foci proximal to hypoxia ( C ) are shown. One 40 µm scale bar is provided for guidance and magnifications correspond to areas in yellow-dotted lines for staining I and II. ( D ) HIF-1α and CAIX protein expression was measured in DLD-1 cells pre-exposed to 1% pO2 (Hx, hypoxia) or 21% pO2 (No, normoxia) for 18 h followed by a 24 hour-infection with EnAd, Ad11p, and Ad5 at MOI 5. ( E ) With the same experimental setup, the down-regulation of HIF signalling was confirmed in HCT116, SW480 and A549 cells infected with EnAd at MOI 5. The corresponding unprocessed western blot images can be found in .

Article Snippet: Primary antibodies were anti-HIF-1α (clone 54, BD Biosciences), anti-HIF-2α (clone D9E3), VHL (polyclonal, Cell Signalling, Beverly, MA, US), anti-CAIX (clone M75, Biosciences Slovakia, Bratislava, Slovakia), and anti-β-Actin antibody (clone AC-74, Sigma Aldrich, Gillingham, UK).

Techniques: Infection, Expressing, In Vitro, Staining, Western Blot

Journal: Cancers

Article Title: Attenuation of the Hypoxia Inducible Factor Pathway after Oncolytic Adenovirus Infection Coincides with Decreased Vessel Perfusion

doi: 10.3390/cancers12040851

Figure Lengend Snippet: Down-regulation of HIF-1α and HIF-2α is independent of proteasomal, prolyl-hydroxylases (PHD) and von-Hippel–Lindau (VHL) activity but dependent on mRNA availability. ( A ) Schematic of the HIF-pathway indicating targets for chemical inhibition. ( B ) DLD-1 cells were pre-exposed to hypoxia (blue) or normoxia (red) for 18 h and infected with EnAd at a multiplicity of infection (MOI) of 5. The infection continued in hypoxia or normoxia for a further 24 h before western blot analysis. Proteasomal inhibition was achieved by adding 5 µM MG-132 six hours before harvest. ( C , D ) Inhibition of VHL was achieved by the silencing of VHL with siRNA transfection 48 h before infection, or VH298 treatment (100 µM) eight hours before harvest. ( E ) PHD inhibition was achieved by adding 75 µM FG-4592 two hours after infection. ( F ) Using the same hypoxia exposure, the mRNA expression of HIF-1α, HIF-2α, GLUT1 and VEGF was measured by RT-qPCR 24 h after infection (m: mock, +: infected, n = 3, ANOVA with Tukey post hoc test, ns: not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). The corresponding unprocessed western blot images can be found in .

Article Snippet: Primary antibodies were anti-HIF-1α (clone 54, BD Biosciences), anti-HIF-2α (clone D9E3), VHL (polyclonal, Cell Signalling, Beverly, MA, US), anti-CAIX (clone M75, Biosciences Slovakia, Bratislava, Slovakia), and anti-β-Actin antibody (clone AC-74, Sigma Aldrich, Gillingham, UK).

Techniques: Activity Assay, Inhibition, Infection, Western Blot, Transfection, Expressing, Quantitative RT-PCR

Journal: Cancers

Article Title: Attenuation of the Hypoxia Inducible Factor Pathway after Oncolytic Adenovirus Infection Coincides with Decreased Vessel Perfusion

doi: 10.3390/cancers12040851

Figure Lengend Snippet: Adenovirus serotype 11p and adenovirus serotype 5 suppress mRNA levels of HIF-1α and HIF-target genes. DLD-1 cells were pre-exposed to hypoxia or normoxia for 18 h and infected with Ad11p ( A ) and Ad5 ( B ) at MOI of 5. The infection continued in hypoxia (blue) or normoxia (red) for a further 24 h. RT-qPCR was performed to measure HIF-1α, HIF-2α, GLUT1 and VEGF (m: mock, +: infected, three biological replicates, ANOVA with Tukey post hoc test, ns: not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: Primary antibodies were anti-HIF-1α (clone 54, BD Biosciences), anti-HIF-2α (clone D9E3), VHL (polyclonal, Cell Signalling, Beverly, MA, US), anti-CAIX (clone M75, Biosciences Slovakia, Bratislava, Slovakia), and anti-β-Actin antibody (clone AC-74, Sigma Aldrich, Gillingham, UK).

Techniques: Infection, Quantitative RT-PCR

Journal: Cancers

Article Title: Attenuation of the Hypoxia Inducible Factor Pathway after Oncolytic Adenovirus Infection Coincides with Decreased Vessel Perfusion

doi: 10.3390/cancers12040851

Figure Lengend Snippet: HIF expression is transiently up-regulated early and down-regulated late during viral infection. ( A ) DLD-1 cells and ( B ) HCT116 cells were pre-exposed for 18 h in normoxia (red) or hypoxia (blue) before infection with EnAd at MOI 5. The infection continued in hypoxia or normoxia for a further eight or 24 h when lysates were collected and probed for HIF-1α, HIF-2α, HIF-1β, CAIX and β-Actin by western blot. Using the same experimental setup, total RNA was collected at the eight hours post-infection from ( C ) DLD-1 and ( D ) HCT116 cells. RT-qPCR was performed to measure HIF-1α , HIF-2α , GLUT1 and VEGF (m: mock, +: infected; n = 3; ANOVA with Tukey post hoc test, ns: not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). The corresponding unprocessed western blot images can be found in .

Article Snippet: Primary antibodies were anti-HIF-1α (clone 54, BD Biosciences), anti-HIF-2α (clone D9E3), VHL (polyclonal, Cell Signalling, Beverly, MA, US), anti-CAIX (clone M75, Biosciences Slovakia, Bratislava, Slovakia), and anti-β-Actin antibody (clone AC-74, Sigma Aldrich, Gillingham, UK).

Techniques: Expressing, Infection, Western Blot, Quantitative RT-PCR

Journal: Cancers

Article Title: Attenuation of the Hypoxia Inducible Factor Pathway after Oncolytic Adenovirus Infection Coincides with Decreased Vessel Perfusion

doi: 10.3390/cancers12040851

Figure Lengend Snippet: Primer sequences, concentration and PCR efficiencies.

Article Snippet: Primary antibodies were anti-HIF-1α (clone 54, BD Biosciences), anti-HIF-2α (clone D9E3), VHL (polyclonal, Cell Signalling, Beverly, MA, US), anti-CAIX (clone M75, Biosciences Slovakia, Bratislava, Slovakia), and anti-β-Actin antibody (clone AC-74, Sigma Aldrich, Gillingham, UK).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Differential expression and function of CAIX and CAXII in breast cancer: A comparison between tumorgraft models and cells

doi: 10.1371/journal.pone.0199476

Figure Lengend Snippet: Panel A . mRNA from all breast cancer patients (unrestricted analysis) was probed for the CA9 gene expression (CAIX-mRNA) using Affimetrix ID 205199_at. Panel B . mRNA expression in triple negative breast cancer patients was probed for CAIX mRNA using Affimetrix ID, 205199. Panel C . mRNA from all breast cancer patients (unrestricted analysis) was probed for the CA12 gene expression (CAXII mRNA) using Affimetrix ID, 215867_at. Panel D . mRNA from all ER-positive breast cancer patients was probed for the CAXII mRNA using Affimetrix ID, 215867_at.

Article Snippet: Membranes were incubated with specific antibodies: CAIX (M75 monoclonal, gift from Dr. Egbert Oosterwijk), CAII (Novus, #NB600-919), CAXII (R&D, # AF2190), estrogen receptor (Santa Cruz, # sc-8002), E-cadherin (BD biosciences, #610181), GAPDH (Cell Signaling, #5174), and actin (Abcam, #ab8226) followed by incubation with horseradish peroxidase–conjugated secondary antibodies (Sigma-Aldrich).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Differential expression and function of CAIX and CAXII in breast cancer: A comparison between tumorgraft models and cells

doi: 10.1371/journal.pone.0199476

Figure Lengend Snippet: Panel A. Frozen tissue samples from PDX tumors were homogenized in RIPA buffer containing protease inhibitor. Western blots were probed with antibodies for CAIX, CAXII, CAII, ER, E-cadherin, actin, and GAPDH. Panel B. Tumor growth rates of orthotopically implanted, cryo-preserved tumor tissue was evaluated in NOD/SCID mice. Panel C. Immunohistochemistry was utilized to evaluate expression of CAIX, CAXII, ER, and Ki67 in TNBC (HCl-001) and ER/PR-positive (HCl-011) tumors from Panel B. Magnification: primary objective magnification, 10x.

Article Snippet: Membranes were incubated with specific antibodies: CAIX (M75 monoclonal, gift from Dr. Egbert Oosterwijk), CAII (Novus, #NB600-919), CAXII (R&D, # AF2190), estrogen receptor (Santa Cruz, # sc-8002), E-cadherin (BD biosciences, #610181), GAPDH (Cell Signaling, #5174), and actin (Abcam, #ab8226) followed by incubation with horseradish peroxidase–conjugated secondary antibodies (Sigma-Aldrich).

Techniques: Protease Inhibitor, Western Blot, Immunohistochemistry, Expressing

Journal: PLoS ONE

Article Title: Differential expression and function of CAIX and CAXII in breast cancer: A comparison between tumorgraft models and cells

doi: 10.1371/journal.pone.0199476

Figure Lengend Snippet: Cell growth was monitored by the MTT assay in which CAIX and CAXII were knocked-down in UFH-001 and T47D cells using shRNA or activity was blocked using the impermeant sulfonamide inhibitor, N-3500 (1mM). Panels A and B. UFH-001 cells. Panels C and D. T47D cells. Asterisks denote statistical significance (* p < 0.05, *** p < 0.001.

Article Snippet: Membranes were incubated with specific antibodies: CAIX (M75 monoclonal, gift from Dr. Egbert Oosterwijk), CAII (Novus, #NB600-919), CAXII (R&D, # AF2190), estrogen receptor (Santa Cruz, # sc-8002), E-cadherin (BD biosciences, #610181), GAPDH (Cell Signaling, #5174), and actin (Abcam, #ab8226) followed by incubation with horseradish peroxidase–conjugated secondary antibodies (Sigma-Aldrich).

Techniques: MTT Assay, shRNA, Activity Assay

Journal: PLoS ONE

Article Title: Differential expression and function of CAIX and CAXII in breast cancer: A comparison between tumorgraft models and cells

doi: 10.1371/journal.pone.0199476

Figure Lengend Snippet: Cell migration and invasion were determined using trans-well chambers. Panel A. UFH-001 cells (empty vector and CRISPR-CAIX knockdown cells from ) were plated in the upper transwell chambers and allowed to migrate or invade across the membrane for 24 h (upper images) or 48 h (lower images), respectively. Tabulation of results is shown to the right ( p < 0.05). Panel B. T47D cells (empty vector or CAXII knockdown cells from ) were plated in the upper transwell chambers and allowed to migrate or invade across the membrane for 24 h (upper images) or 48 h (lower images), respectively. Tabulation of results is shown to the right.

Article Snippet: Membranes were incubated with specific antibodies: CAIX (M75 monoclonal, gift from Dr. Egbert Oosterwijk), CAII (Novus, #NB600-919), CAXII (R&D, # AF2190), estrogen receptor (Santa Cruz, # sc-8002), E-cadherin (BD biosciences, #610181), GAPDH (Cell Signaling, #5174), and actin (Abcam, #ab8226) followed by incubation with horseradish peroxidase–conjugated secondary antibodies (Sigma-Aldrich).

Techniques: Migration, Plasmid Preparation, CRISPR

Journal: PLoS ONE

Article Title: Differential expression and function of CAIX and CAXII in breast cancer: A comparison between tumorgraft models and cells

doi: 10.1371/journal.pone.0199476

Figure Lengend Snippet: 18 O exchange was used to measure CA activity in cells at pH 6.8, 7.4, and 7.9. Panels A and B. A representative plot of CAIX activity in response to pH is shown for UFH-001 cells exposed to normoxic or hypoxic conditions. Three independent biological replicates were used to quantify the first order rate constants. Panels C and D. A representative plot of CAXII activity in response to pH is shown for T47D cells exposed to normoxic and hypoxic conditions. Three independent biological replicates were used to quantify the first order rate constants. The asterisks denote statistical significance. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Membranes were incubated with specific antibodies: CAIX (M75 monoclonal, gift from Dr. Egbert Oosterwijk), CAII (Novus, #NB600-919), CAXII (R&D, # AF2190), estrogen receptor (Santa Cruz, # sc-8002), E-cadherin (BD biosciences, #610181), GAPDH (Cell Signaling, #5174), and actin (Abcam, #ab8226) followed by incubation with horseradish peroxidase–conjugated secondary antibodies (Sigma-Aldrich).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Differential expression and function of CAIX and CAXII in breast cancer: A comparison between tumorgraft models and cells

doi: 10.1371/journal.pone.0199476

Figure Lengend Snippet: Membrane ghosts were prepared from hypoxic UFH-001 and normoxic T47D cells. Panel A. 18 O exchange data is reported as the rate of activity divided by the enzyme concentration in a pH-dependent manner. The concentration of CAIX in the membrane suspension was estimated at 4.1 nM. The concentration of CAXII in the membrane suspension was estimated at 31.4 nM. Panel B. The kcat exch /K eff s (kcat/Km) with units, M -1 s -1 , is shown for the hydration of CO 2 and dehydration of bicarbonate catalyzed by CAIX. Panel C. the kcat exch /K eff s (kcat/Km) with units, M -1 s -1 , is shown for the hydration of CO 2 and dehydration of bicarbonate catalyzed by CAXII.

Article Snippet: Membranes were incubated with specific antibodies: CAIX (M75 monoclonal, gift from Dr. Egbert Oosterwijk), CAII (Novus, #NB600-919), CAXII (R&D, # AF2190), estrogen receptor (Santa Cruz, # sc-8002), E-cadherin (BD biosciences, #610181), GAPDH (Cell Signaling, #5174), and actin (Abcam, #ab8226) followed by incubation with horseradish peroxidase–conjugated secondary antibodies (Sigma-Aldrich).

Techniques: Activity Assay, Concentration Assay