Journal: Journal of Endocrinological Investigation

Article Title: Carbonic anhydrases III and IX are new players in the crosstalk between adrenocortical carcinoma and its altered adipose microenvironment

doi: 10.1007/s40618-023-02008-4

Figure Lengend Snippet: CAIII and CAIX expression in the adipose tissue. A Representative images of immunofluorescence staining were obtained from both SAT and VAT samples showing CAIII and CAIX expression in both lean and obese patients as red and green signals, respectively. Of note, the higher dimension of the adipocytes in AT from obese vs. lean subjects suggest pathological differences. DAPI staining (blue) was used to mark cell nuclei. Scale bar = 50 µm. B Western blot analysis of CAIII (left panel) and CAIX (right panel) protein expression in SAT and VAT samples from lean and obese patients. The upper panels show a representative blot for CAIII (left) and CAIX (right), while the graphs show the mean ± SE expression levels of CAs normalized to total protein loading and in fold increase vs. SAT of lean patient. * P < 0.05; ** P < 0.001 SAT vs. VAT. °° P < 0.001 lean vs. obese. C qRT-PCR gene expression of CA3 (left panel) and CA9 (right panel) normalized on GAPDH as the reference gene. Data are expressed as the mean ± SE performed in triplicates in at least two independent experiments. * P < 0.05, ** P < 0.001 SAT vs. VAT, ° P < 0.05, °° P < 0.001 lean vs. obese ( n = 4 lean and n = 8 obese subjects)

Article Snippet: mRNA isolated from tumor and adipose tissues or from cell cultures as detailed previously [ , ] were subjected to quantitative real-time RT-PCR (qRT-PCR) for the following genes: CA3 , CA9 , ADIPOQ (adiponectin) , FABP4 (fatty acid binding protein 4) and GAPDH genes (Taqman Gene Expression Assay, Life Technologies, respective codes: Hs00193123_m1, Hs00154208_m1, Hs00605917_m1, Hs00609791_m1, FAM-MGB 4325934–1301038).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Gene Expression

Journal: Journal of Endocrinological Investigation

Article Title: Carbonic anhydrases III and IX are new players in the crosstalk between adrenocortical carcinoma and its altered adipose microenvironment

doi: 10.1007/s40618-023-02008-4

Figure Lengend Snippet: CAIII and CAIX expression in ACC samples. A Representative images of both hematoxylin/eosin (upper panel) and immunofluorescence (middle and bottom panels) staining were obtained in serial slices of healthy adrenal specimens (NOR), stage I ACC and advanced stage IV ACC. The expression of CAIII and CAIX is shown in adrenal cortex area as a red signal. DAPI was used for nuclear staining. Scale bar = 100 µm, 20 µm. B qRT-PCR gene expression of CA3 (left panel) and CA9 (right panel) normalized on GAPDH as the reference gene in n = 23 ACC and n = 2 healthy adrenal specimens (NOR). Data are expressed as mean ± SE performed in triplicates in at least two independent experiments. * P < 0.05; ** P < 0.001 stage I–II ( n = 9) vs. stage III–IV ( n = 14)

Article Snippet: mRNA isolated from tumor and adipose tissues or from cell cultures as detailed previously [ , ] were subjected to quantitative real-time RT-PCR (qRT-PCR) for the following genes: CA3 , CA9 , ADIPOQ (adiponectin) , FABP4 (fatty acid binding protein 4) and GAPDH genes (Taqman Gene Expression Assay, Life Technologies, respective codes: Hs00193123_m1, Hs00154208_m1, Hs00605917_m1, Hs00609791_m1, FAM-MGB 4325934–1301038).

Techniques: Expressing, Immunofluorescence, Staining, Quantitative RT-PCR, Gene Expression

Journal: Journal of Endocrinological Investigation

Article Title: Carbonic anhydrases III and IX are new players in the crosstalk between adrenocortical carcinoma and its altered adipose microenvironment

doi: 10.1007/s40618-023-02008-4

Figure Lengend Snippet: Modulation of CAIII and CAIX expression in adipose and tumor cells under coculturing conditions. A CA3 and CA9 gene expression analysis performed by qRT-PCR in adipose stem cells (ASC), in vitro-differentiated adipocytes (Adipo), H295R and MUC-1 cells. Data are expressed as mean ± SE gene expression vs. the housekeeping GAPDH gene. § P < 0.05, §§ P < 0.001 ASC vs. Adipo and H295R vs. MUC-1. B qRT-PCR gene expression analysis of CA3 and CA9 in adrenocortical cancer cells lines in monoculture and in coculture conditions with ASCs. Data are expressed as mean ± SE gene expression normalized on GAPDH as the reference gene and in fold increase of coculturing conditions vs. tumor cells alone taken as 1 (dotted line). qRT-PCR gene expression analysis of CA3 C and CA9 D evaluated in adipose cells in monoculture and in coculture conditions with H295R or MUC-1 cells. ASCs cultured in undifferentiating conditions (ASC) or under stimulation toward adipogenesis (Adipo), alone or in the presence of tumor cells. Data are expressed as mean ± SE gene expression normalized on GAPDH as the reference gene and in fold increase of coculturing conditions vs. adipose cells (ASCs or adipocytes, respectively) alone taken as 1 (dotted line). * P < 0.01 cocultured conditions vs. alone; # P < 0.05 cocultured adipo vs. cocultured ASCs

Article Snippet: mRNA isolated from tumor and adipose tissues or from cell cultures as detailed previously [ , ] were subjected to quantitative real-time RT-PCR (qRT-PCR) for the following genes: CA3 , CA9 , ADIPOQ (adiponectin) , FABP4 (fatty acid binding protein 4) and GAPDH genes (Taqman Gene Expression Assay, Life Technologies, respective codes: Hs00193123_m1, Hs00154208_m1, Hs00605917_m1, Hs00609791_m1, FAM-MGB 4325934–1301038).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, In Vitro, Cell Culture

Journal: American journal of physiology. Renal physiology

Article Title: Studies of ultrastructure, gene expression, and marker analysis reveal that mouse bladder PDGFRA + interstitial cells are fibroblasts.

doi: 10.1152/ajprenal.00135.2022

Figure Lengend Snippet: Figure 3. Distribution of mouse bladder Pdgfra/PDGFRA þ fibroblasts. A: expression of Pdgfra in the bladder wall of PdgfraH2B-EGFP reporter mice as revealed by confocal microscopy. Tissue was counterstained with phalloidin to reveal the actin cytoskeleton (white) and DAPI (blue) to reveal cell nuclei. Actin was associated with the cortical cytoskeleton of most cells but strongly labeled the actin filaments that abound in the cytoplasm of smooth muscle cells. As expected for a nucleus-localized reporter, the H2B-enhanced green fluorescent protein (EGFP) signal overlapped completely with the DAPI sig- nal (see insets for example of the DAPI signal alone and EGFP signal alone). Representative confocal micrographs from two mice are shown. B: bladder tissue from PdgfraH2B-EGFP reporter mice was counterstained with antibodies to PDGFRA, CAR3, or CD34. Representative confocal micrographs from two mice are shown. Examples of H2B-EGFP þ nuclei that were also positive for PDGFRA, CAR3, or CD34 are marked with yellow arrows. In the top images, a small number of cells were found that were PDGFRAþ but H2B-EGFP– (nuclei marked with orange arrows) or were H2B-EGFPþ but PDGFRA– (nuclei labeled with cyan arrows). C: PDGFRAþ cells (green) were found in the following regions: the suburothelial region (SU); in the subja- cent outer lamina propria (OLP), which forms the core of the rougae (Ru) and extends below suburothelial cells in the unfolded regions; within the peri- mysial connective tissue in the intermuscular region (IM); and in the serosa (Se), where a single layer of cells was sandwiched between the smooth muscle and mesothelial cell (MC) layer. The tissue was counterstained with DAPI (blue) and phalloidin (white; bottom images). The boxed regions in the top left images are magnified in the adjacent images. Representative confocal micrographs from four experiments (each with 2–3 mice) are shown.

Article Snippet: Primary antibodies used in the present study Antigen Host Species Source Catalog Number Dilution of stock ACTA2 Rabbit Proteintech 14395-1-AP 1:100 CAR3 Rabbit Proteintech 15197-1-AP 1:100 (1:100 for FISHID) CD34 Sheep R&D Systems AF6518 1:100 (1:50 for FISHID) CD34 Rat (clone MEC14.7) Invitrogen (ThermoFisher) MA1-22646 1:200 CSPG4 Rabbit Millipore Sigma AB5320 1:100 GFP Rabbit Abcam Ab6556 1:75 for EM KIT (CD117) Rat (clone ACK2) Stemcell 60034.1 1:100 KRT20 Rabbit Abcam ab53120 1:100 KRT5 Chicken BioLegend 905901 1:400 LY6A Rat BioLegend 108101 1:100 MYH11 Rabbit Proteintech 21404-1-AP 1:100 (1:100 for FISHID) PDGFRA Goat R&D Systems AF1062 1:100 (1:50 for FISHID) Preferred antibody used in most experiments PECAM1 (CD31) Rat (clone 390) Millipore Sigma CBL1337-I 1:100 PI16 Goat R&D Systems AF4929-SP 1:100 PI16 Rabbit US Biological 141796 1:100 PTPRC (CD45) Rat (clone IBL-3/16) Bio-Rad MCA1388 1:100 TNC Rat (clone no. 578) R&D Systems MAB2138 1:400 UCHL1 (PGP9.5) Rabbit Genetex GTX109637 1:100 VIM Rat BioLegend 699301 1:100 FISHID, fluorescent in situ hybridization with immunodetection.

Techniques: Expressing, Confocal Microscopy, Labeling

Journal: American journal of physiology. Renal physiology

Article Title: Studies of ultrastructure, gene expression, and marker analysis reveal that mouse bladder PDGFRA + interstitial cells are fibroblasts.

doi: 10.1152/ajprenal.00135.2022

Figure Lengend Snippet: Figure 6. PDGFRAþ cells express Col1a2. A: expression of Col1a2-driven mG (mem- brane-bound enhanced green fluorescent protein) in tamoxifen-treated experimental Col1a2Cre-ERT þ /–;RosamT/mG þ /– or control Col1a2Cre-ERT–/–;RosamT/mG þ /– mice. B: coexpression of mG in the PDGFRAþ, CAR3 þ, or CD34þ cells of the indicated regions of the bladder wall [suburothelial (SU), outer lamina propria (OLP), intermus- cular (IM), and serosal (Se)]. Examples of cells with visible nuclei coexpressing these markers are marked with yellow arrows. Coexpression of MYH11 and mG was also assessed in the bottom right images. Examples of smooth muscle cells expressing mG are indicated by white arrowheads. C: use of fluorescent in situ hybridization with immunodetection (FISHID) to assess coexpression of PDGFRA, Pdgfra, and Col1a2 in the indicated regions of the bladder wall. Examples of cells (i.e., nuclei) that expressed all three markers are labeled with white arrowheads; cells that were PDGFRAþ;Pdgfraþ are marked with green arrowheads. The image marked “Control” was performed in the absence of anti- PDGFRA antibody and using the three-plex negative control probe against Bacillus sub- tilis dapB. All images are confocal micro- graphs and representative of images taken from two separate experiments each using two mice. BV, blood vessels; MC, mesothe- lial cells; Ut, urothelium.

Article Snippet: Primary antibodies used in the present study Antigen Host Species Source Catalog Number Dilution of stock ACTA2 Rabbit Proteintech 14395-1-AP 1:100 CAR3 Rabbit Proteintech 15197-1-AP 1:100 (1:100 for FISHID) CD34 Sheep R&D Systems AF6518 1:100 (1:50 for FISHID) CD34 Rat (clone MEC14.7) Invitrogen (ThermoFisher) MA1-22646 1:200 CSPG4 Rabbit Millipore Sigma AB5320 1:100 GFP Rabbit Abcam Ab6556 1:75 for EM KIT (CD117) Rat (clone ACK2) Stemcell 60034.1 1:100 KRT20 Rabbit Abcam ab53120 1:100 KRT5 Chicken BioLegend 905901 1:400 LY6A Rat BioLegend 108101 1:100 MYH11 Rabbit Proteintech 21404-1-AP 1:100 (1:100 for FISHID) PDGFRA Goat R&D Systems AF1062 1:100 (1:50 for FISHID) Preferred antibody used in most experiments PECAM1 (CD31) Rat (clone 390) Millipore Sigma CBL1337-I 1:100 PI16 Goat R&D Systems AF4929-SP 1:100 PI16 Rabbit US Biological 141796 1:100 PTPRC (CD45) Rat (clone IBL-3/16) Bio-Rad MCA1388 1:100 TNC Rat (clone no. 578) R&D Systems MAB2138 1:400 UCHL1 (PGP9.5) Rabbit Genetex GTX109637 1:100 VIM Rat BioLegend 699301 1:100 FISHID, fluorescent in situ hybridization with immunodetection.

Techniques: Expressing, Control, In Situ Hybridization, Immunodetection, Labeling, Negative Control

Journal: American journal of physiology. Renal physiology

Article Title: Studies of ultrastructure, gene expression, and marker analysis reveal that mouse bladder PDGFRA + interstitial cells are fibroblasts.

doi: 10.1152/ajprenal.00135.2022

Figure Lengend Snippet: Figure 9. PDGFRAþ suburothelial fibroblasts express ACTA2, CAR3, and TNC. A: ACTA2 was expressed by smooth muscle cells (SMC) in the muscularis externa and blood vessels (BV) within the lamina propria. A weaker, but reproducible, ACTA2 signal was also observed in suburo- thelial fibroblasts, subjacent to the urothelium (Ut). The dashed boxed region in the top right image is magnified in the bottom images. Nuclei of PDGFRA þ;ACTA2 þ cells are indicated by yellow arrows. B: expression of TNC and CAR3 by PDGFRAþ suburothelial fibroblasts. The dashed boxed region in the right image is reproduced in the left images. Cells with visible nuclei expressing all three markers are indicated by white arrows. Note that PDGFRA is a mem- brane protein, CAR3 is a cytoplasmic protein, and TNC is a secreted protein that is deposited in the space between ad- jacent fibroblasts. All images are confocal micrographs and representative of images taken from three-five separate experiments each with one-two mice.

Article Snippet: Primary antibodies used in the present study Antigen Host Species Source Catalog Number Dilution of stock ACTA2 Rabbit Proteintech 14395-1-AP 1:100 CAR3 Rabbit Proteintech 15197-1-AP 1:100 (1:100 for FISHID) CD34 Sheep R&D Systems AF6518 1:100 (1:50 for FISHID) CD34 Rat (clone MEC14.7) Invitrogen (ThermoFisher) MA1-22646 1:200 CSPG4 Rabbit Millipore Sigma AB5320 1:100 GFP Rabbit Abcam Ab6556 1:75 for EM KIT (CD117) Rat (clone ACK2) Stemcell 60034.1 1:100 KRT20 Rabbit Abcam ab53120 1:100 KRT5 Chicken BioLegend 905901 1:400 LY6A Rat BioLegend 108101 1:100 MYH11 Rabbit Proteintech 21404-1-AP 1:100 (1:100 for FISHID) PDGFRA Goat R&D Systems AF1062 1:100 (1:50 for FISHID) Preferred antibody used in most experiments PECAM1 (CD31) Rat (clone 390) Millipore Sigma CBL1337-I 1:100 PI16 Goat R&D Systems AF4929-SP 1:100 PI16 Rabbit US Biological 141796 1:100 PTPRC (CD45) Rat (clone IBL-3/16) Bio-Rad MCA1388 1:100 TNC Rat (clone no. 578) R&D Systems MAB2138 1:400 UCHL1 (PGP9.5) Rabbit Genetex GTX109637 1:100 VIM Rat BioLegend 699301 1:100 FISHID, fluorescent in situ hybridization with immunodetection.

Techniques: Expressing

Journal: PLoS ONE

Article Title: High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

doi: 10.1371/journal.pone.0067546

Figure Lengend Snippet: List of top 20 most highly expressed genes in human corneal endothelium.

Article Snippet: CA3 , Hs01013316_m1 , 4331182 , .

Techniques: Migration, Binding Assay, Membrane, Transduction, Activation Assay, Control, Cell Culture, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

doi: 10.1371/journal.pone.0067546

Figure Lengend Snippet: GO analysis using DAVID Functional Annotation (Subset: GOTERM_BP_FAT).

Article Snippet: CA3 , Hs01013316_m1 , 4331182 , .

Techniques: Functional Assay, Phospho-proteomics

Journal: PLoS ONE

Article Title: High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

doi: 10.1371/journal.pone.0067546

Figure Lengend Snippet: List of top 50 highly expressed genes found in human corneal endothelium that are 2-fold higher in CECs than in stroma, and less than 2-fold difference between young and old corneal endothelium.

Article Snippet: CA3 , Hs01013316_m1 , 4331182 , .

Techniques:

Journal: PLoS ONE

Article Title: High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

doi: 10.1371/journal.pone.0067546

Figure Lengend Snippet: Primer list.

Article Snippet: CA3 , Hs01013316_m1 , 4331182 , .

Techniques:

Journal: Annals of palliative medicine

Article Title: Carbonic anhydrase 2 and 3 as risk biomarkers for dilated cardiomyopathy associated heart failure.

doi: 10.21037/apm-21-3561

Figure Lengend Snippet: Figure 3 CA2 and CA3 may serve as risk biomarkers for DCM of HF. (A) The folding change levels of the top 15 raised proteins and the

Article Snippet: The contents of the CA2 and CA3 in the plasma were measured by human CA2 and CA3 ELISA kits (CUSABIO; CSB-E08823h, CSB-E15962h; Wuhan, China) in accordance with the manufacturer’s standard protocol.

Techniques: