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

Journal: American Journal of Physiology - Renal Physiology

Article Title: Highly tamoxifen-inducible principal cell-specific Cre mice with complete fidelity in cell specificity and no leakiness

doi: 10.1152/ajprenal.00436.2017

Figure Lengend Snippet: Aqp2ECE is inactive in intercalated cells marked by carbonic anhydrase II (CAII). A–C: immunofluorescence staining for Aqp2 (blue) and CAII (green) to mark intercalated cells and RFP (red) showing no RFP coexpression with CAII in whole kidney of adult ECE/+RFP/RFP mice treated with a single dose of tamoxifen (2 mg) for 24 h. Cells indicated by arrowheads are magnified in the insets. Scale bar, 50 μm, 8.3 μm for inset.

Article Snippet: Two mouse antibodies specific for carbonic anhydrase II (CAII, sc-48351) and V-ATPase B1 B2 (sc-55544), one rabbit Aqp2 antibody (sc-28629), and two goat antibodies against Aqp2 (sc-9882) and Pendrin (sc-16894) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Immunofluorescence, Staining