Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A) Amino acid sequence of the human CAIX proteoglycan-like domain. Negatively charged amino acids are labelled in red, positively charged amino acids are labelled in blue. (B, C) Original recordings of the change in intracellular H + concentration ([H + ] i ) in oocytes expressing (B) MCT1 or (C) MCT4 (black trace), MCT1/4 + CAIX-WT (blue trace), and MCT1/4 + CAIX-ΔPG (red trace), respectively, during application of 3 and 10 mM of lactate and 5% CO 2 / 10 mM HCO 3 - . (D-I) Rate of change in intracellular H + concentration (Δ[H + ]/Δt) as induced by application (D, G) and removal (E, H) of 3 and 10 mM lactate, and application of 5% CO 2 / 10 mM HCO 3 - (F, I), respectively, in oocytes expressing MCT1/4 (gray), MCT1/4+CAIX-WT (blue), and MCT1+CAII-ΔPG (red), respectively. Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars for MCT1/4+CAIX/CAIX-ΔPG-coexpressing oocytes refer to the values from oocytes expressing MCT1/4 alone. The blue significance indicators above the bars for MCT1/4+CAIX-ΔPG-coexpressing oocytes refer to the values from oocytes coexpressing MCT1/4 + CAIX.

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Sequencing, Concentration Assay, Expressing, Comparison

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: Antibody staining of fixed and permeabilized oocytes, expressing MCT1+CAIX-WT (A) , MCT4+CAIX-WT (B) , MCT1+CAIX-ΔPG (C) , MCT4+CAIX-ΔPG (D) , and a native oocyte as control (E) . CAIX was labeled with an antibody, mapping against the C-terminal region of CAIX. Pictures were taken with a confocal laser scanning microscope.

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Staining, Expressing, Control, Labeling, Laser-Scanning Microscopy

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A, B) Original recordings of the change in intracellular H + concentration ([H + ] i ) in oocytes coexpressing MCT1+CAIX-WT (A) and MCT4+CAIX (B), respectively, during application of 3 and 10 mM lactate. Cells were pre-incubated for 24 h with 0.4 μg/ml Anti-PG (red traces), 0.4 μg/ml Anti-CA (green traces) or without antibodies (black traces) before the measurements were carried out. (C, D) Rate of change in intracellular H + concentration (Δ[H + ]/Δt) as induced by application (C, E) and removal (D, F) of 3 and 10 mM lactate, in oocytes expressing MCT1/4 (gray) and MCT1/4+CAIX-WT (blue), respectively, in the presence of Anti-PG, Anti-CA or in the absence of antibodies (control). Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars for MCT1/4+CAIX-coexpressing oocytes refer to the corresponding values from oocytes expressing MCT1/4 alone (gray bars). The blue significance indicators above the bars for MCT1/4+CAIX-coexpressing oocytes, incubated with Anti-PG or Anti-CA refer to the values from MCT1/4+CAIX-coexpressing oocytes not incubated with antibody (blue bars). No significant changes were found between MCT1/4-expressing oocytes incubated with or without antibodies (gray bars).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Concentration Assay, Incubation, Expressing, Control, Comparison

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A) Original recordings of the log enrichment (LE), as measured by gas-analysis mass spectrometry, of a pool of 20 lysed, native oocytes (black trace) and pools of 20 lysed oocytes, expressing either CAIX-WT (blue trace) or CAIX-ΔPG (red trace). The beginning of the traces shows the rate of degradation of the 18 O-labeled substrate in the non-catalyzed reaction. The black arrowhead indicates addition of oocyte lysate. (B) Enzymatic activity of lysates from 20 native oocytes (gray) and 20 oocytes expressing either CAIX-WT (blue) or CAIX-ΔPG (red), respectively. One unit is defined as 100% stimulation of the non-catalyzed 18 O depletion of doubly labelled 13 C 18 O 2 . Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from CAIX-expressing oocytes refer to the values from native cells (gray bar). The blue asterisks above the bar from CAIX-ΔPG-expressing oocytes refer to the values from CAIX-WT-expressing oocytes (blue bar). (C) Enzymatic activity of cell lysate from 20 native (gray) and CAIX-expressing oocytes (blue, red and green), respectively. Cell lysates were incubated for 2h with 5 μg/ml of Anti-PG (red), 5 μg/ml of Anti-CA (green), or without antibody (blue). One unit is defined as 100% stimulation of the non-catalyzed 18 O depletion of doubly labelled 13 C 18 O 2 . Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from CAIX-expressing oocytes refer to the values from native cells (gray bar). The blue asterisks above the bar from CAIX-expressing oocytes, incubated with antibodies refer to the values from CAIX-WT-expressing oocytes without antibody (blue bar). The red asterisks above the bar from CAIX-expressing oocytes, incubated with Anti-CA refer to the values from CAIX-WT-expressing oocytes, incubated with Anti-PG (red bar).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Mass Spectrometry, Expressing, Labeling, Activity Assay, Comparison, Incubation

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A) Original recordings of the change in intracellular pH (pH i ) during application of 3 and 10 mM of lactate in MCF-7 breast cancer cells, incubated under normoxia (left traces) or hypoxia (right traces), in the presence of 5 μg/ml Anti-PG (red traces), 5 μg/ml Anti-CA (green traces) or in the absence of antibodies (black traces). (B, C) Rate of change in intracellular pH, as induced by application (B) and removal (C) of lactate in MCF-7 cells, incubated under normoxia (gray) or hypoxia (blue), in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (D) Relative change in intracellular lactate concentration, as induced by application of lactate in MCF-7 cells, incubated under normoxia (gray) or hypoxia (blue) in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). Data are represented as mean ± SEM. Significance in differences was tested with ANOVA, followed by means comparison. The significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (Ctrl). (E) CA catalytic activity of MCF-7 cells, as measured by gas-analysis mass spectrometry, in the presence 5 μg/ml Anti-PG (red), 5 μg/ml Anti-CA (green), 30 μM of the CA inhibitor EZA (yellow) and in the absence of antibodies or inhibitors (Ctrl). Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black asterisks above the bars from cells treated with antibodies or EZA refer to the values from control cells (gray bar). The red asterisks above the bars from cells treated with Anti-CA or EZA refer to the values from cells treated with Anti-PG (red bar). The green significance indicator above the bar from cells treated with EZA refers to the values from cells treated with Anti-CA (green bar). (F) Western blot against CAIX and β-tubulin as loading control, in MCF-7 cells, incubated in the presence Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (G) Quantification of the protein level of CAIX in MCF-7 cells, incubated in the presence Anti-PG (red), Anti-CA (green) or in the absence of antibodies (gray) relative to the protein level of β-tubulin. Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (gray bar). The red significance indicator above the bar from cells treated with Anti-CA refers to the values from cells treated with Anti-PG (red bar).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Incubation, Concentration Assay, Comparison, Control, Activity Assay, Mass Spectrometry, Western Blot

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A) Original recordings of the change in intracellular pH (pH i ) during application of 3 and 10 mM of lactate, in MDA-MB-231 breast cancer cells, incubated under normoxia (left traces) or hypoxia (right traces), in the presence of 5 μg/ml Anti-PG (red traces), 5 μg/ml Anti-CA (green traces) or in the absence of antibodies (black traces). (B, C) Rate of change in intracellular pH, as induced by application (B) and removal (C) of lactate in MDA-MB-231 cells, incubated under normoxia (gray) or hypoxia (blue), in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (D) Relative change in intracellular lactate concentration, as induced by application of lactate in MDA-MB-231 cells, incubated under normoxia (gray) or hypoxia (blue) in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). Data are represented as mean ± SEM. Significance in differences was tested with ANOVA, followed by means comparison. The significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (Ctrl). (E) CA catalytic activity of MDA-MB-231 cells, as measured by gas-analysis mass spectrometry, in the presence 5 μg/ml Anti-PG (red), 5 μg/ml Anti-CA (green), 30 μM of the CA inhibitor EZA (yellow) and in the absence of antibodies or inhibitors (Ctrl). Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black asterisks above the bars from cells treated with antibodies or EZA refer to the values from control cells (gray bar). The red asterisks above the bars from cells treated with Anti-CA or EZA refer to the values from cells treated with Anti-PG (red bar). The green significance indicator above the bar from cells treated with EZA refers to the values from cells treated with Anti-CA (green bar). (F) Western blot against CAIX and β-tubulin as loading control, in MDA-MB-231 cells, incubated in the presence Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (G) Quantification of the protein level of CAIX in MDA-MB-231 cells, incubated in the presence Anti-PG (red), Anti-CA (green) or in the absence of antibodies (gray) relative to the protein level of β-tubulin. Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (gray bar). The red significance indicator above the bar from cells treated with Anti-CA refers to the values from cells treated with Anti-PG (red bar).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Incubation, Concentration Assay, Comparison, Control, Activity Assay, Mass Spectrometry, Western Blot

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A) Western blot of CAIX, in normoxic and hypoxic MCF-7 and MDA-MB-231 cells with Anti-PG (Ab-M75, upper blots). β-tubulin was used as loading control (lower blots). (B) Quantification of the protein level of CAIX in normoxic and hypoxic MDA-MB-231 and MCF-7 cells, respectively, relative to the protein level of β-tubulin. Data are represented as mean + SEM. Significance in differences was tested with Student's t-test, following a Shapiro-Wilk test. The significance indicators above the bars for normoxic and hypoxic MCF-7 cells refer to the corresponding values for normoxic and hypoxic MDA-MB-231 cells (gray bars).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Western Blot, Control

Journal: Advanced Science

Article Title: Transgelin Promotes Glioblastoma Stem Cell Hypoxic Responses and Maintenance Through p53 Acetylation

doi: 10.1002/advs.202305620

Figure Lengend Snippet: Preferential expression of TAGLN in hypoxia‐induced GSCs. A) Volcano plot of differentially expressed genes at peritumoral regions and pseudopalisades in GBM tissue. Red dots, genes upregulated in hypoxia; Blue dots, genes downregulated in hypoxia. B) Representative images of HE (Scale bar, 100 µm) and IHC (Scale bar, 50 µm) stained in pseudopalisades located around the necrosis (upper) and other areas (lower) of serial sections of hGBM samples. The red arrow points to the necrosis (N) in GBM and the black arrow represents the pseudopalisades (P) around the necrosis. The large black square in the lower‐left corner of the image is an enlarged view of the small black square in the image. C) Representative images of IF staining for TAGLN, CA9, and HIF1α in hGBM tissues. Scale bar, 50 µm. D) Expression levels of TAGLN and HIF1α were examined by IB in GSCs cultured in 1% O 2 for the indicated time. E) Representative images of IF staining for TAGLN and putative GSCs markers in hGBM sections. Scale bar, 25µm. F) Cell lysates were analyzed by IB for TAGLN, OLIG2, and GFAP expression. G) The expression of TAGLN in GSCs and glioma cells was tested by IB. H) IB analyzed TAGLN expression in GSCs and NPCs. I) Kaplan‐Meier curve showing patient survival based on HIF1A mRNA expression in the CGGA GBM (IDH‐WT) dataset. J) The Kaplan‐Meier survival curve was calculated to measure survival in the group of TAGLN hi HIF1A hi ( n = 139), TAGLN hi HIF1A lo , ( n = 73), TAGLN lo HIF1A hi , ( n = 73), and TAGLN lo HIF1A lo ( n = 139).

Article Snippet: IHC was performed using antibodies against anti‐TAGLN (Cat# ab14106, RRID:AB_443021, Abcam), anti‐SOX2 (Cat# ab171380, RRID:AB_2732072, Abcam), anti‐HIF1α (Cat# 66730‐1‐Ig, RRID: AB_288208, Proteintech), anti‐CA9 (Cat# NB100‐417, RRID:AB_10003398, NOVUS), anti‐GFAP (Cat# 80788, RRID:AB_2799963, Cell Signaling Technology), anti‐Ki67(Cat# 27309‐1‐AP, RRID:AB_2756525, Proteintech), anti‐Cleaved Caspase3 (Cat# 9664, RRID:AB_2070042, Cell Signaling), anti‐HDAC2 (Cat# ab219053, Abcam), anti‐Acetyl‐p53(K382) (Cat# 2525, RRID:AB_330083, Cell Signaling Technology), anti‐p21 (Cat# 2947, RRID:AB_823586, Cell Signaling Technology), anti‐CCNE1 (Cat# ET1612‐16, HUABIO), anti‐CDK2 (Cat# YT0832, Immunoway biotechnology), anti‐CCND1 (Cat# 26939‐1‐AP, RRID:AB_2880691, Proteintech), anti‐p27 (Cat# 3686, RRID:AB_2077850, Cell Signaling Technology), and anti‐CDK4 (Cat# 12790, RRID:AB_2631166, Cell Signaling Technology).

Techniques: Expressing, Staining, Cell Culture

Journal: Advanced Science

Article Title: Transgelin Promotes Glioblastoma Stem Cell Hypoxic Responses and Maintenance Through p53 Acetylation

doi: 10.1002/advs.202305620

Figure Lengend Snippet: TAGLN binds to HDAC2 to form a co‐transcriptional activator complex. A) Expression of HDAC2 and TAGLN was detected by IB. B) Expression of TAGLN and HDAC2 in GSCs after HDAC2 knockdown was examined by IB. C) Expression of HDAC2, TAGLN, and HIF1α during HIF1α targeting was detected by IB under hypoxia. D) Representative IHC images of TAGLN, HDAC2, CA9, and HIF1α expression in the xenografts. Scale bar, 50 µm. E) IOD of HDAC2, TAGLN, CA9, and HIF1α were measured in (D). F) IB analysis of TAGLN and HDAC2 in different treatment groups. G,H) IF staining of HDAC2 (red) and LAMP1 (lysosomal marker; green) after DMSO or chloroquine treatment of TAGLN targeted GSCs. Scale bar, 10 µm. I) IHC staining revealed HDAC2, HIF1α, CA9, and TAGLN expression in pseudopalisades (upper) or other areas (lower) of GBM. Scale bar,50 µm. J) Representative images of TAGLN and HDAC2 expression in GBM specimens. Scale bar, 25 µm. K) Co‐IP analysis of TAGLN and HDAC2 in three GSC cell lines. Values in (E) represent the mean ± SD from three independent experiments (* p < 0.05, **p < 0.01, and ***p < 0.001).

Article Snippet: IHC was performed using antibodies against anti‐TAGLN (Cat# ab14106, RRID:AB_443021, Abcam), anti‐SOX2 (Cat# ab171380, RRID:AB_2732072, Abcam), anti‐HIF1α (Cat# 66730‐1‐Ig, RRID: AB_288208, Proteintech), anti‐CA9 (Cat# NB100‐417, RRID:AB_10003398, NOVUS), anti‐GFAP (Cat# 80788, RRID:AB_2799963, Cell Signaling Technology), anti‐Ki67(Cat# 27309‐1‐AP, RRID:AB_2756525, Proteintech), anti‐Cleaved Caspase3 (Cat# 9664, RRID:AB_2070042, Cell Signaling), anti‐HDAC2 (Cat# ab219053, Abcam), anti‐Acetyl‐p53(K382) (Cat# 2525, RRID:AB_330083, Cell Signaling Technology), anti‐p21 (Cat# 2947, RRID:AB_823586, Cell Signaling Technology), anti‐CCNE1 (Cat# ET1612‐16, HUABIO), anti‐CDK2 (Cat# YT0832, Immunoway biotechnology), anti‐CCND1 (Cat# 26939‐1‐AP, RRID:AB_2880691, Proteintech), anti‐p27 (Cat# 3686, RRID:AB_2077850, Cell Signaling Technology), and anti‐CDK4 (Cat# 12790, RRID:AB_2631166, Cell Signaling Technology).

Techniques: Expressing, Staining, Marker, Immunohistochemistry, Co-Immunoprecipitation Assay

Journal: Medical Oncology (Northwood, London, England)

Article Title: Expression of HIF-1α and CAIX in nasopharyngeal carcinoma and their correlation with patients’ prognosis

doi: 10.1007/s12032-014-0304-1

Figure Lengend Snippet: Expression of HIF-1α and CAIX by immunohistochemistry in NPC tissues (original magnification ×400). a Nuclear membrane positive staining of HIF-1α in NPC tissue. b Plasmalemma and cytoplasmic positive staining of CAIX in NPC tissue

Article Snippet: Each section was incubated with protein blocker for 15 min at room temperature followed by mouse anti-human HIF-1α antibody(1:100, Novus Biologicals, Colorado, USA) and rabbit anti-human CAIX antibody (1:400, Novus Biologicals, Colorado, USA)incubation at 4 °C overnight.

Techniques: Expressing, Immunohistochemistry, Membrane, Staining

Journal: Medical Oncology (Northwood, London, England)

Article Title: Expression of HIF-1α and CAIX in nasopharyngeal carcinoma and their correlation with patients’ prognosis

doi: 10.1007/s12032-014-0304-1

Figure Lengend Snippet: The Kaplan–Meier curves for NPC patients with both positive and negative expressions of HIF-1α and CAIX: a overall survival, b progression-free survival, c loco-regional relapse-free survival, d distant metastasis-free survival

Article Snippet: Each section was incubated with protein blocker for 15 min at room temperature followed by mouse anti-human HIF-1α antibody(1:100, Novus Biologicals, Colorado, USA) and rabbit anti-human CAIX antibody (1:400, Novus Biologicals, Colorado, USA)incubation at 4 °C overnight.

Techniques:

Journal: British Journal of Cancer

Article Title: High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer

doi: 10.1038/sj.bjc.6605690

Figure Lengend Snippet: Correlation of clinicopathological parameters with the CAIX expression level detected by immunohistochemistry in 555 NSCLC patients

Article Snippet: Briefly, a rabbit polyclonal anti-CAIX antibody (clone ab15086, diluted 1 : 2000, Abcam, Cambridge, MA, USA) was added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with a biotinylated mouse anti-rabbit IgG (Abcam) as the secondary antibody.

Techniques: Expressing, Immunohistochemistry

Journal: British Journal of Cancer

Article Title: High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer

doi: 10.1038/sj.bjc.6605690

Figure Lengend Snippet: Correlation of clinicopathological parameters with the CAIX plasma level detected by ELISA in 209 NSCLC patients

Article Snippet: Briefly, a rabbit polyclonal anti-CAIX antibody (clone ab15086, diluted 1 : 2000, Abcam, Cambridge, MA, USA) was added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with a biotinylated mouse anti-rabbit IgG (Abcam) as the secondary antibody.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: British Journal of Cancer

Article Title: High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer

doi: 10.1038/sj.bjc.6605690

Figure Lengend Snippet: CAIX expression detected by immunohistochemistry in tissue microarray cores. Staining levels for CAIX in NSCLC histological subtypes: low ( A , E , I , M ), intermediate ( B , F , J , N ) and strong ( C , G , K , O ). CAIX membrane staining in adenocarcinoma (ADC) ( A – D ), squamous cell carcinoma (SCC) ( E – H ), large cell carcinoma (LCC) ( I – L ), and sarcomatoid carcinoma (SC) ( M – P ). Normal bronchial epithelium ( Q ) and alveolar tissue ( R ) are devoid of staining. S trong membrane staining in head and neck squamous cell carcinoma (HNSCC) cores used as positive control of CAIX immunostaining ( S – T ). Panels D , H , L , P and T are higher magnifications showing details of cells within the corresponding tumour shown on panels C , G , K , O , and S , respectively.

Article Snippet: Briefly, a rabbit polyclonal anti-CAIX antibody (clone ab15086, diluted 1 : 2000, Abcam, Cambridge, MA, USA) was added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with a biotinylated mouse anti-rabbit IgG (Abcam) as the secondary antibody.

Techniques: Expressing, Immunohistochemistry, Microarray, Staining, Membrane, Positive Control, Immunostaining

Journal: British Journal of Cancer

Article Title: High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer

doi: 10.1038/sj.bjc.6605690

Figure Lengend Snippet: Quantitative evaluation of the plasma CAIX level by ELISA in patients with NSCLC and in healthy controls. ( A ) Box plots showing median (central dots), 25–75% quartile ranges (boxes), and minimum/maximum levels (whiskers) of plasma CAIX levels in healthy individuals ( n =58) vs patients with NSCLC ( n =209). ( B ) ROC curve analysis of CAIX as a plasma marker for NSCLC. X axis, 1-specificity; y axis, sensitivity. ( C ) Plasma concentration of CAIX according to tumour size in NSCLC and in healthy individuals. NS; non significant.

Article Snippet: Briefly, a rabbit polyclonal anti-CAIX antibody (clone ab15086, diluted 1 : 2000, Abcam, Cambridge, MA, USA) was added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with a biotinylated mouse anti-rabbit IgG (Abcam) as the secondary antibody.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Marker, Concentration Assay

Journal: British Journal of Cancer

Article Title: High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer

doi: 10.1038/sj.bjc.6605690

Figure Lengend Snippet: Kaplan–Meier curves of overall survival (top panels A and C ), and disease-specific survival (bottom panels B and D ) duration stratified according to the tissue CAIX expression detected by immunohistochemistry (left panels A and B ) and the plasma CAIX levels as determined by ELISA (right panels C and D ). The cut-off value for the CAIX immunostaining score was arbitrarily defined as superior or equal to 45 grey levels. The cut-off point for the CAIX plasma levels was empirically defined as superior or equal to 100 pg ml –1 . The curves are labelled with the corresponding scores.

Article Snippet: Briefly, a rabbit polyclonal anti-CAIX antibody (clone ab15086, diluted 1 : 2000, Abcam, Cambridge, MA, USA) was added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with a biotinylated mouse anti-rabbit IgG (Abcam) as the secondary antibody.

Techniques: Expressing, Immunohistochemistry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Immunostaining

Journal: British Journal of Cancer

Article Title: High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer

doi: 10.1038/sj.bjc.6605690

Figure Lengend Snippet: Multivariate Cox regression analysis of tumour tissue and plasma CAIX levels expression for OS and DSS in patients with NSCLC

Article Snippet: Briefly, a rabbit polyclonal anti-CAIX antibody (clone ab15086, diluted 1 : 2000, Abcam, Cambridge, MA, USA) was added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with a biotinylated mouse anti-rabbit IgG (Abcam) as the secondary antibody.

Techniques: Clinical Proteomics, Expressing

Journal: British Journal of Cancer

Article Title: Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours

doi: 10.1038/sj.bjc.6605200

Figure Lengend Snippet: Immunofluorescence analysis performed on ( A – C , P – U ) human colorectal adenocarcinoma LS174T, ( D – F ) human colorectal adenomacarcinoma SW1222, ( G – I ) human colorectal adenocarcinoma HT-29, ( J – L ) human glioblastoma U87 and ( M – O ) human RCC SK-RC-52 xenografted tumour tissue sections. Red staining in the left panels represents endothelial cells (anti-CD31 staining), whereas green staining in the middle panels represents expression of CA IX (( B , E , H , K , N ) SIP(A3) or ( T ) polyclonal anti-CA IX antiserum staining). Overlay of red and green fluorescence is shown in the panels to the right. ( Q ) Primary antibody was omitted as a negative control. Scale bar=100 μ m.

Article Snippet: Three serial sections from the same tumour were incubated with a 1:2 dilution of rat anti-mouse CD31 simultaneously with one of the following antibodies: (1) 1 : 200 dilution of rabbit anti-pimonidazole (Natural Pharmacia International Inc.), (2) 1 : 1000 dilution of rabbit anti-human IgE (Dako; to detect the injected SIP(A3) and SIP(CC7)) or (3) 1 : 10 dilution of rabbit polyclonal anti-CAIX antiserum (sc-25599; Santa Cruz).

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, Negative Control

Journal: British Journal of Cancer

Article Title: Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours

doi: 10.1038/sj.bjc.6605200

Figure Lengend Snippet: Multi-fluorescence microscopy analysis in LS174T xenograft-bearing mice. ( A , B ) Representative overlays of multiple digital fluorescence images of a LS174T tumour injected with pimonidazole (30 min before killing) and SIP(A3) (6 h before killing) demonstrating perfusion with Hoechst 33342 (1 min before killing) (blue), blood vessel staining (red), pimonidazole binding (green, left panel) and CA IX targeting by SIP(A3) (green, middle panel). ( C ) ex vivo staining of an adjacent tumour section with a polyclonal anti-CA IX antiserum. ( D – F ) Higher magnification images of mainly perfused areas of the corresponding tumour sections. ( G – I ) Higher magnification images of mainly CA IX-positive areas of the corresponding tumour sections. ( A – C ) Scale bar=500 μ m and ( D – I ) scale bar=100 μ m. N indicates necrosis.

Article Snippet: Three serial sections from the same tumour were incubated with a 1:2 dilution of rat anti-mouse CD31 simultaneously with one of the following antibodies: (1) 1 : 200 dilution of rabbit anti-pimonidazole (Natural Pharmacia International Inc.), (2) 1 : 1000 dilution of rabbit anti-human IgE (Dako; to detect the injected SIP(A3) and SIP(CC7)) or (3) 1 : 10 dilution of rabbit polyclonal anti-CAIX antiserum (sc-25599; Santa Cruz).

Techniques: Fluorescence, Microscopy, Injection, Staining, Binding Assay, Ex Vivo

Journal: British Journal of Cancer

Article Title: Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours

doi: 10.1038/sj.bjc.6605200

Figure Lengend Snippet: Multi-fluorescence microscopy analysis in SW1222 xenograft-bearing mice. ( A and B ) Representative overlays of multiple digital fluorescence images of a SW1222 tumour injected with pimonidazole (30 min before killing) and SIP(A3) (6 h before killing) demonstrating perfusion with Hoechst 33342 (1 min before killing) (blue), blood vessel staining (red), pimonidazole binding (green, left panel) and CA IX targeting by SIP(A3) (green, middle panel). ( C ) ex vivo staining of an adjacent tumour section with a polyclonal anti-CA IX antiserum. ( D – F ) Higher magnification images of mainly perfused areas of the corresponding tumour sections. ( G – I ) Higher magnification images of mainly CA IX-positive areas of the corresponding tumour sections. ( A – C ) Scale bar=500 μ m and ( D – I ) scale bar=100 μ m.

Article Snippet: Three serial sections from the same tumour were incubated with a 1:2 dilution of rat anti-mouse CD31 simultaneously with one of the following antibodies: (1) 1 : 200 dilution of rabbit anti-pimonidazole (Natural Pharmacia International Inc.), (2) 1 : 1000 dilution of rabbit anti-human IgE (Dako; to detect the injected SIP(A3) and SIP(CC7)) or (3) 1 : 10 dilution of rabbit polyclonal anti-CAIX antiserum (sc-25599; Santa Cruz).

Techniques: Fluorescence, Microscopy, Injection, Staining, Binding Assay, Ex Vivo

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: The CAIX antibodies (goat anti-mouse CAIX AF2344, goat anti-human CAIX AF2188, monoclonal mouse anti-human CAIX MAB2188) and the mouse anti-IgG 2A (MAB003) isotype control were obtained from R&D systems (Minneapolis, MN, USA).

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

Journal: Journal of the American Heart Association

Article Title: Endothelial p53 Deletion Improves Angiogenesis and Prevents Cardiac Fibrosis and Heart Failure Induced by Pressure Overload in Mice

doi: 10.1161/jaha.115.001770

Figure Lengend Snippet: Figure 8. Cardiac vascularization in End.p53-WT and End.p53-KO mice. A, Representative pictures of CD31-immunopositive, isolectin B4-perfused capillaries in hearts of sham- or TAC-operated End.p53-WT and End.p53-KO mice. B, Quantification of CD31-positive cells per mm2. C, Quantification of the lectin- positive area. D, Immunostaining for CAIX to visualize cardiac hypoxia. Size bars represent 100 lm. E, The summary of the quantitative analysis in n=4 to 9 mice per group. *P<0.05 and ***P<0.001 vs sham. Significant differences between End.p53-WT and End.p53-KO mice are indicated within the graphs. TAC indicates transverse aortic constriction.

Article Snippet: A rabbit anti-carbonic anhydrase IX (CAIX) antibody (Bioss Antibodies) was used as surrogate marker for hypoxia.23 Fibroblast-specific protein (FSP)-1 was detected using a polyclonal rabbit anti-mouse antibody (Dako Cytomation).

Techniques: Immunostaining