Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 and stromal-interacting molecule 1 (STIM1) expression in mouse pulmonary arterial smooth muscle cells (PASMCs). A: RT-PCR products from cultured mouse PASMCs amplified using primers for mouse Orai1 (269 bp), STIM1 (473 bp), and β-actin (498 bp). Three separate RT-PCR reactions were performed in the presence (+) and absence (−) of reverse transcriptase (RT). B: Orai1, STIM1, and GAPDH proteins were detected in cultured mouse PASMCs using Western blot analysis. Experiments were performed in 5 separate Western blot analyses.

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Amplification, Reverse Transcription, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 mediates capacitative Ca2+ entry (CCE) in mouse PASMCs. A and B: Orai1 protein and GAPDH were detected in nontransfected mouse PASMCs and in PASMCs transfected with 200 nM scrambled small interfering RNA (siRNA; negative control). The expression of Orai1 but not GAPDH was reduced significantly in cells transfected with 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: siRNA knockdown of Orai1 reduced the cyclopiazonic acid (CPA)-induced transient but not the sustained increase in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. 0Ca, Ca2+-free solution. D: bar graph showing mean changes in transient and sustained increase in intracellular Ca2+ concentration ([Ca2+]i) caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in negative control cells (filled bars, n = 89), and in Orai1 siRNA-transfected cells (open bars, n = 86). **P < 0.01 (unpaired t-test). E: siRNA knockdown of Orai1 reduced the increase in Mn2+ quench of fura-2 fluorescence caused by 10 μM CPA in the presence of 10 μM nifedipine. AU, arbitrary units. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in negative control cells (filled bar, n = 79), and in Orai1 siRNA-transfected cells (open bar, n = 86). **P < 0.01 (unpaired t-test).

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Transfection, Small Interfering RNA, Negative Control, Expressing, Western Blot, Knockdown, Fluorescence, Concentration Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: STIM1 is associated with Orai1 to mediate CCE in mouse PASMCs. A and B: STIM1, Orai1, and GAPDH proteins were detected in nontransfected mouse PASMCs and in PASMCs transfected with 200 nM scrambled siRNA (negative control). The expression of STIM1 but not Orai1 or GAPDH was reduced significantly in cells transfected with 200 nM STIM1 siRNA. The expressions of STIM1 and Orai1 but not GAPDH were reduced significantly in cells transfected with both 200 nM STIM1 siRNA and 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: siRNA knockdown of STIM1 reduced the CPA-induced transient and sustained increase in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. siRNA knockdown of STIM1 and Orai1 further reduced the CPA-induced transient but not sustained increase in fura-2 fluorescence ratio in the presence of nifedipine. D: bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in negative control cells (filled bars, n = 103), in STIM1 siRNA-transfected cells (shaded bars, n = 148), and in STIM1 siRNA and Orai1 siRNA-transfected cells (open bars, n = 70). **P < 0.01, compared with negative control cells (ANOVA); ++P < 0.01, compared with negative control cells and STIM1 siRNA-transfected cells (ANOVA). E: siRNA knockdown of STIM1 reduced the increase in Mn2+ quench of fura-2 fluorescence caused by 10 μM CPA in the presence of 10 μM nifedipine. siRNA knockdown of STIM1 and Orai1 further reduced the increase in Mn2+ quench of fura-2 fluorescence caused by CPA in the presence of nifedipine. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in negative control cells (filled bar, n = 125), in STIM1 siRNA-transfected cells (shaded bar, n = 151), and in STIM1 siRNA and Orai1 siRNA-transfected cells (open bar, n = 137). **P < 0.01, compared with negative control cells (ANOVA); ++P < 0.01, compared with the negative control cells and STIM1 siRNA-transfected cells (ANOVA).

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Transfection, Negative Control, Expressing, Western Blot, Knockdown, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: knockdown of Orai1 reduced CCE in STIM1-overexpressing mouse PASMCs. A and B: STIM1, Orai1, and GAPDH proteins were detected in cells infected with adenovirus containing green fluorescent protein (Ad-GFP) transfected with 200 nM scrambled siRNA. The expression of STIM1 but not Orai1 or GAPDH increased markedly in cells infected with STIM1-GFP-adenovirus (Ad-GFP-STIM1) transfected with scrambled siRNA. The expression of Orai1 but not STIM1 or GAPDH was reduced significantly in STIM1-overexpressing cells transfected with 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: overexpression of STIM1 in scrambled siRNA-transfected cells caused an increase in CPA-induced transient and sustained rise in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. The increases in fluorescence ratio were reduced in Orai1 siRNA-transfected cells overexpressed with STIM1. D: bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in GFP-infected cells transfected with scrambled siRNA (filled bars, n = 33), in STIM1-overexpressing cells transfected with scrambled siRNA (shaded bars, n = 65), and in STIM1-overexpressing cells transfected with Orai1 siRNA (open bars, n = 50). *P < 0.05, compared with GFP-infected cells transfected with scrambled siRNA and STIM1-overexpressing cells transfected with Orai1 siRNA (ANOVA). E: overexpression of STIM1 in scrambled siRNA-transfected cells caused a CPA-induced increase in Mn2+ quench of fura-2 fluorescence in the presence of 10 μM nifedipine. The increases in Mn2+ quench of fura-2 fluorescence was reduced in Orai1 siRNA-transfected cells overexpressed with STIM1. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in GFP-infected cells transfected with scrambled siRNA (filled bars, n = 40), in STIM1-overexpressing cells transfected with scrambled siRNA (shaded bars, n = 95), and in STIM1-overexpressing cells transfected with Orai1 siRNA (open bars, n = 57). **P < 0.01, compared with GFP-infected cells transfected with scrambled siRNA and STIM1-overexpressing cells transfected with Orai1 siRNA (ANOVA).

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Knockdown, Infection, Transfection, Expressing, Western Blot, Over Expression, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 coimmunoprecipitates with STIM1 in mouse PASMCs. A, left: Orai1 was detected in cultured mouse PASMCs in the absence and presence of store-depletion. Right: bar graph showing expression levels of Orai1 measured relative to GAPDH in control cells (denoted as 1, filled bar) and in cells subjected to store-depletion (open bar). Data are means ± SE of 7 separate Western blot analyses. B, left: STIM1 was detected in cultured mouse PASMCs in the absence and presence of store-depletion. Right: bar graph showing expression levels of STIM1 measured relative to GAPDH in control cells (denoted as 1, filled bar) and in cells subjected to store-depletion (open bar). Data are means ± SE of 7 separate Western blot analyses. C: STIM1 coimmunoprecipitated Orai1 in cultured mouse PASMCs in the absence and presence of store-depletion. STIM1 was first immunoprecipitated (IP) with EXBIO STIM1 antibody (10 μg), and the blot was subsequently probed with BD Biosciences STIM1 antibody (WB, 1:100). The blot was then probed for coimmunoprecipitation (co-IP) of Orai1 expression using Orai1 antibody (WB, 1:100, ProSci). Experiments were performed in 3 separate co-IP procedures and Western blot analyses.

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Cell Culture, Expressing, Control, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Colocalization of Orai1 and STIM1 in mouse PASMCs. A–D: staining of mouse cultured PASMCs after exposure of live cells with normal bath physiological salt solution (PSS). A: omission of Orai1 and STIM1 antibody resulted in no Orai1 or STIM1 staining. B–D: three representative cells dual-labeled with anti-Orai1 antibody (green) and STIM1 antibody (red). Orai1 and STIM1 colocalization (yellow/orange) is shown in the merged images. E–H: staining of mouse cultured PASMCs after exposure of live cells with Ca2+-free PSS containing 10 μM CPA. E: omission of Orai1 and STIM1 antibody resulted in no Orai1 or STIM1 staining. F–H: three representative cells dual-labeled with anti-Orai1 antibody (green) and STIM1 antibody (red). Colocalization of Orai1 and STIM1 is more apparent (yellow/orange) after store-depletion as shown in the merged images. Nuclei were stained with DAPI (blue). Experiments were performed in 3 separate immunostaining procedure, each with duplicate coverslips. Scale bars, 20 μm.

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Staining, Cell Culture, Labeling, Immunostaining

Journal: Circulation

Article Title: HNRNPA2B1: an RNA-binding protein that orchestrates smooth muscle cell phenotype in pulmonary arterial hypertension

doi: 10.1161/CIRCULATIONAHA.122.059591

Figure Lengend Snippet: ( A ) Localization and quantification of A2B1 protein in PASMC within the lung of healthy and PAH subjects (A2B1 in red, α-SMA in white, Nuclei in blue). Quantification of A2B1 ( B ) protein and ( C ) mRNA in primary cultured PASMC isolated from healthy and PAH subjects. ( D ) Quantification of A2B1 expression from online available PAH-PASMC RNA-sequencing ( GSE144274 ). ( E ) Localization of A2B1 in primary cultured PASMC assessed by nucleo-cytoplasmic separation showing the increased nuclear expression of A2B1 in PAH-PASMC compared to Ctrl-PASMC. ( F ) Localization of A2B1 in primary cultured PASMC assessed by immunofluorescence showing an accumulation of A2B1 in the nuclei in PAH-PASMC compared to Ctrl-PASMC. The histogram represents the fluorescence intensity throughout the measured area (dotted white line). Dapi is shown in the blue spectrum and A2B1 in the red spectrum. ( G ) Alignment of both isoform of A2B1 (HNRNPA2 -NM002137.4 - and HNRNPB1 – NM031243.3) together showing the only sequence mismatch between them. These 36 nucleotides that are absent from the A2 isoform are annotated in the uniprot database to contain a nuclear localization. The alignment was performed using Clustal Omega. ( H ) mRNA expression of the isoform B1 in PAH-PASMC compared to Ctrl-PASMC.

Article Snippet: Ctrl PASMC were purchased from ATCC (PCS-100–023) and PAH-PASMC were isolated in our laboratory.

Techniques: Expressing, Cell Culture, Isolation, RNA Sequencing, Immunofluorescence, Fluorescence, Sequencing

Journal: Circulation

Article Title: HNRNPA2B1: an RNA-binding protein that orchestrates smooth muscle cell phenotype in pulmonary arterial hypertension

doi: 10.1161/CIRCULATIONAHA.122.059591

Figure Lengend Snippet: Panel ( A ) summarizes our analysis pipeline to identify the mRNA targets of A2B1 in human and its function in PAH-PASMC. ( B ) To define the putative targets of A2B1 we used the A2B1 motifs sequences (ATtRACT database) and search for them on mRNA sequences (UTRs and CCDS databases). We identified three motifs with enough complexity to be accurately detected and all mRNAs containing these motifs which are the putative targets of A2B1. We also found that these three motifs were enriched in 3’UTRs region. ( C ) To apply this knowledge in PAH, we identified all putative targets of A2B1 in an RNA-sequencing of primary cultured Ctrl vs PAH PASMC. ( D ) To define the subset of mRNAs actively regulated by A2B1 in PAH-PASMC, we correlated the expression of A2B1 with its putative targets. From this correlation one clear pattern emerged – a subset of significantly dysregulated putative targets of A2B1 was positively correlated with A2B1 (R > 0.7) in PAH-PASMC – These targets are the A2B1 targets in PAH-PASMC. The density plots shows the percent of putative targets of A2B1 in function of the Spearman correlation coefficient (R) ( E ) To define the function of A2B1 in PAH-PASMC we performed a gene ontology enrichment analysis using the A2B1 targets for each motif. We found that most of the enriched gene ontology term were related to regulation of the cell cycle regulation. The GOchord graph shows the link between each motif and the enriched gene ontology term associated.

Article Snippet: Ctrl PASMC were purchased from ATCC (PCS-100–023) and PAH-PASMC were isolated in our laboratory.

Techniques: Expressing, RNA Sequencing, Cell Culture

Journal: Circulation

Article Title: HNRNPA2B1: an RNA-binding protein that orchestrates smooth muscle cell phenotype in pulmonary arterial hypertension

doi: 10.1161/CIRCULATIONAHA.122.059591

Figure Lengend Snippet: Panel ( A ), ( B ) and ( C ) respectively shows for the yellow motif, the blue motif and the red motif, the correlation between A2B1 representative targets and A2B1 in RNA-seq (purple) and RT-qPCR (green) on the left panel. In addition, the right panels shows, respectively, the expression of the representative targets in RNA-seq and RT-qPCR and the results of an RNA-immunoprecipitation (RNA-IP) demonstrating the up-regulation of each target in PAH-PASMC vs Ctrl-PASMC and their binding to A2B1 in PAH-PASMC. ( D ) quantification of Ki-67-positive PASMC (red) to assess proliferation and ( E ) quantification of cleaved caspase 3 positive PASMC to assess apoptosis (red) in PASMC Ctrl vs PAH.

Article Snippet: Ctrl PASMC were purchased from ATCC (PCS-100–023) and PAH-PASMC were isolated in our laboratory.

Techniques: Biomarker Discovery, Cell Culture, RNA Sequencing, Quantitative RT-PCR, Expressing, RNA Immunoprecipitation, Binding Assay

Journal: Circulation

Article Title: HNRNPA2B1: an RNA-binding protein that orchestrates smooth muscle cell phenotype in pulmonary arterial hypertension

doi: 10.1161/CIRCULATIONAHA.122.059591

Figure Lengend Snippet: ( A ) A2B1 and isoform B1 mRNA expression. ( B ) A2B1 protein expression in PAH-PASMC treated with Si-Scrm or Si-A2B1. ( C ) Gene set enrichment analysis of RNA-sequencing comparing PAH-PASMC vs Ctrl-PASMC. This analysis revealed that as a set of targets, the targets of A2B1 as well as all the pathways we found to be regulated by A2B1 in Fig.2 were significantly down-regulated when A2B1 was silenced. ( D ) mRNA expression of A2B1 selected targets demonstrating their down-regulation in PAH-PASMC treated with Si-A2B1. In addition, PAH-PASMC treated with Si-A2B1 demonstrate ( E ) a decrease in proliferation and ( F ) an increase in apoptosis.

Article Snippet: Ctrl PASMC were purchased from ATCC (PCS-100–023) and PAH-PASMC were isolated in our laboratory.

Techniques: Expressing, RNA Sequencing

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Dectin-1 is markedly upregulated in the chronic kidney disease (CKD), presumably by macrophages, and is correlated significantly with declined renal function and progressive renal fibrosis. (A) Representative photomicrographs of Dectin-1 (red) immunofluorescence staining in renal cortical tissue from patients with diabetic kidney disease (DKD), hypertensive nephropathy (HN), lupus nephritis (LN), minimal change disease (MCD), membranous nephropathy (MN), and mesangial proliferative glomerulonephritis (MsPGN). Nuclei stained with DAPI (4',6'-diamidino-2-phenylindole) are shown in blue. Scale bar, 50 μm. (B) Dectin-1 is highly enriched in macrophages, identified from an independent public data: GSE199711 . (C) Representative dual-immunofluorescence staining of Dectin-1 and CD68 in the fibrotic renal tissues of patients with CKD. Scale bar, 50 μm. (D) Representative photomicrographs of Hematoxylin and eosin (H&E) staining, Periodic Acid-Schiff (PAS) staining, Periodic-acid silver methenamine (PASM) staining, and Masson's trichrome staining in IgA nephropathy (IgAN). Scale bar, 50 μm. (E) Representative photomicrographs of Dectin-1 immunofluorescence staining in IgAN (n = 42 for each group). Scale bar, 50 μm. (F) Quantification of the Dectin-1 + cells in (E). (G) The correlation between the number of Dectin-1 + cells and serum creatinine (Scr), blood urea nitrogen (BUN), urinary protein, fibrosis, serum albumin, and estimated glomerular filtration rate (eGFR) in IgAN patients (n = 84). Data are presented as mean ± SD. **** P < 0.0001.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques: Immunofluorescence, Staining, Tissue, Filtration, PASM, Single Cell RNA-Seq

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Logistic regression analysis of various clinicopathological factors and the number of Dectin-1 + cells for renal fibrosis in IgAN patients.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques: Analysis

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Dectin-1 is markedly upregulated in mouse chronic kidney disease (CKD) models induced by unilateral ureteric obstruction (UUO), folic acid (FA), and ischemia-reperfusion (IR) and is presumably expressed by macrophages. (A) Representative photomicrographs illustrating H&E staining in the kidney tissues of mice at day 7 after UUO or sham operation. Scale bar, 50 μm. (B) Representative photomicrographs illustrating H&E staining in the kidney tissues of mice at day 14 after FA induction. Scale bar, 50 μm. (C) Representative photomicrographs illustrating H&E staining in the kidney tissues of mice at day 14 after IR or sham operation. Scale bar, 50 μm. (D) Relative mRNA expression of Clec7a (Dectin-1) in the kidney tissues of mice at day 7 after UUO or sham operation (n = 6 for each group). (E) Relative mRNA expression of Clec7a (Dectin-1) in the kidney tissues of mice at day 14 after FA induction (n = 6 for each group). (F) Relative mRNA expression of Clec7a (Dectin-1) in the kidney tissues of mice at day 14 after IR or sham operation (n = 6 for each group). (G) Representative flow cytometric dot plots (left) and quantifications (2 parts in the right) illustrating the percentage of Dectin-1 + cells and its mean fluorescence intensity in the kidney tissues of mice at day 7 after UUO or sham operation (n=6 for each group). (H) Representative flow cytometric dot plots (left) and quantifications (2 parts in the right) illustrating the percentage of Dectin-1 + cells and its mean fluorescence intensity in the kidney tissues of mice at day 14 after FA induction (n=6 for each group). (I) Representative flow cytometric dot plots (left) and quantifications (2 parts in the right) illustrating the percentage of Dectin-1 + cells and its mean fluorescence intensity in the kidney tissues of mice at day 14 after IR or sham operation (n=6 for each group). (J) Dectin-1 is highly enriched in macrophages, identified from an independent public data: GSE175412 . (K) Representative dual-immunofluorescence staining of Dectin-1 and F4/80 in the fibrotic kidney tissues of mice. Scale bar, 50 μm. Data are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques: Staining, Expressing, Fluorescence, Immunofluorescence, Real-time Polymerase Chain Reaction, FACS, Single Cell RNA-Seq

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Mice lacking Dectin-1 are protected from unilateral ureteric obstruction (UUO)-induced progressive renal fibrosis. Wild-type (WT) and Dectin-1 knockout (KO) mice challenged to sham or UUO operation. (A) Experimental design of the treatment procedure. (B) Representative photomicrographs illustrating H&E and Masson's trichrome staining in kidney tissues. Scale bar, 50 μm. (C) Representative photomicrographs illustrating immunohistochemical staining for fibronectin, collagen1, vimentin, and α-SMA in kidney tissues (n = 6 for each group). Scale bar, 50 μm. (D) Quantification of the positive staining area (%) in (C). (E) Representative western blot analysis of fibronectin, collagen1, vimentin, and α-SMA protein in the kidney tissues. α-Tubulin was used as loading control (n = 6 for each group). (F) Densitometric quantification of blots in (E). (G) Relative mRNA expression of Fn1 , Col1a1 , Vim , and Acta2 in kidney tissues (n = 6 for each group). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques: Knock-Out, Staining, Immunohistochemical staining, Western Blot, Analysis, Control, Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Deletion of bone marrow (BM)-derived Dectin-1 inhibits unilateral ureteric obstruction (UUO)-induced renal fibrosis. (A) Experimental design of the treatment procedure. (B) Flow cytometric analysis showing the genetic background of CD45 alleles in the wild-type (WT) and Dectin-1 knockout (KO) mice. (C) Successful BM transplantations (BMTs) were confirmed by flow cytometric analysis of peripheral blood cells. (D-I) After successful BMT, mice from 4 groups (WT BM→WT, Dectin-1 KO BM→WT, WT BM→ Dectin-1 KO, and Dectin-1 KO BM→ Dectin-1 KO) were subjected to sham or UUO operation. (D) Representative photomicrographs illustrating H&E and Masson's trichrome staining in kidney tissues. Scale bar, 50 μm. (E) Representative photomicrographs illustrating immunohistochemical staining for fibronectin, collagen1, vimentin, and α-SMA in kidney tissues (n=3 for each group). Scale bar, 50 μm. (F) Quantification of the positive staining area (%) in (E). (G) Representative western blot analysis of fibronectin, collagen1, vimentin, and α-SMA protein in the kidney tissues. α-Tubulin was used as loading control (n = 3 for each group). (H) Densitometric quantification of blots in (G). (I) Relative mRNA expression of Fn1 , Col1a1 , Vim , and Acta2 in kidney tissues (n = 3 for each group). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques: Derivative Assay, Analysis, Knock-Out, Staining, Immunohistochemical staining, Western Blot, Control, Expressing, FACS, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Macrophage-specific deletion of Dectin-1 protects against unilateral ureteric obstruction (UUO)-induced renal fibrosis. Dectin-1 f/f and Dectin-1 ΔCd68 mice challenged to sham or UUO operation. (A) Experimental design of the treatment procedure. (B) Representative photomicrographs illustrating H&E and Masson's trichrome staining in kidney tissues. Scale bar, 50 μm. (C) Representative photomicrographs illustrating immunohistochemical staining for fibronectin, collagen1, vimentin, and α-SMA in kidney tissues (n=6 for each group). Scale bar, 50 μm. (D) Quantification of the positive staining area (%) in (C). (E) Representative western blot analysis of fibronectin, collagen1, vimentin, and α-SMA protein in the kidney tissues. α-Tubulin was used as loading control (n = 6 for each group). (F) Densitometric quantification of blots in (E). (G) Relative mRNA expression of Fn1 , Col1a1 , Vim , and Acta2 in kidney tissues (n = 6 for each group). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques: Staining, Immunohistochemical staining, Western Blot, Analysis, Control, Expressing, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Dectin-1 deficiency suppresses macrophage accumulation and activation in fibrotic kidneys. Wild-type (WT) and Dectin-1 knockout (KO) mice subjected to sham or unilateral ureteric obstruction (UUO) operation. (A-D) Representative flow cytometric dot plots (left) and quantification (right) showing percentages of leukocytes (CD45 + live cells), macrophages (CD45 + CD11b + F4/80 + live cells), neutrophils (CD45 + CD11b + Ly6G + live cells) and dendritic cells (CD45 + CD11c + MHC II + live cells) in kidneys (n=6 for each group). (E) Representative photomicrographs illustrating immunohistochemical staining for F4/80 in kidney tissues (n=6 for each group). Scale bar, 50 μm. (F) Quantification of the positive staining area (%) in (E). (G) Representative flow cytometric images illustrating the expression of iNOS + in CD45 + CD11b + F4/80 + live cells (left) and the percentage of iNOS + cells in macrophages and its mean fluorescence intensity levels (2 parts in the right) in kidney tissues 7 days after UUO (n=6 for each group). (H) Representative flow cytometric images illustrating the expression of CD206 + in CD45 + CD11b + F4/80 + live cells (left) and the percentage of CD206 + cells in macrophages and its mean fluorescence intensity levels (2 parts in the right) in kidney tissues 7 days after UUO (n=6 for each group). Data are presented as mean ± SD. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques: Activation Assay, Knock-Out, Immunohistochemical staining, Staining, Expressing, Fluorescence, FACS, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Dectin-1 deletion reduces macrophage chemotaxis through CCL2 (C-C motif chemokine ligand 2)-CCR2 (C-C motif chemokine receptor 2) axis. Wild-type (WT) and Dectin-1 knockout (KO) mice subjected to unilateral ureteric obstruction (UUO) operation. (A) Representative western blot analysis of CCL2 protein in the kidney tissues. α-Tubulin was used as loading control (n = 6 for each group). (B) Densitometric quantification of blots in (A). (C) Representative flow cytometric images illustrating the expression of CCR2 + in CD11b + F4/80 + live cells (left) and the percentage of CCR2 + cells in macrophages and its mean fluorescence intensity levels (2 parts in the right) in kidneys (n=6 for each group). (D) Relative mRNA expression of Ccr2 in sorted macrophages from fibrotic kidneys (n = 5 for each group). (E) Relative mRNA expression of Ccl2 in sorted macrophages from fibrotic kidneys (n = 5 for each group). (F) The concentrations of CCL2 detected by ELISA in supernatant from sorted macrophages of fibrotic kidneys (n = 5 for each group). (G) Schematic of Dectin-1 signaling. (H) Representative flow cytometric images illustrating the expression of phospho (p-) spleen tyrosine kinase (Syk) + in CD45 + CD11b + F4/80 + live cells (left) and the percentage of p-Syk + cells in macrophages and its mean fluorescence intensity levels (2 parts in the right) in kidneys (n=6 for each group). (I) Representative western blot analysis of p-IκBα, IκBα, p-p65, and p65 protein in the kidney tissues. α-Tubulin was used as loading control (n = 6 for each group). (J) Densitometric quantification of blots in (I). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques: Chemotaxis Assay, Knock-Out, Western Blot, Analysis, Control, Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay, FACS, Real-time Polymerase Chain Reaction

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Dectin-1 deficiency reduces macrophage-to-myofibroblast transition (MMT) through TGF-β/Smad pathway. Wild-type (WT) and Dectin-1 knockout (KO) mice subjected to unilateral ureteric obstruction (UUO) operation. (A) Representative flow cytometric images illustrating the expression of α-SMA + in CD45 + CD11b + F4/80 + live cells (left) and the percentage of α-SMA + cells in macrophages and its mean fluorescence intensity levels (2 parts in the right) in kidneys (n=6 for each group). (B) Relative mRNA expression of Acta2 in sorted macrophages from fibrotic kidneys (n = 5 for each group). (C) Schematic of Dectin-1 induced MMT. (D) Representative western blot analysis of TGF-β1 protein in the kidney tissues. α-Tubulin was used as loading control (n = 6 for each group). (E) Densitometric quantification of blots in (D). (F) Relative mRNA expression of Tgfb1 in kidney tissues (n = 5 for each group). (G) Relative mRNA expression of Tgfb1 in sorted macrophages from fibrotic kidneys (n = 5 for each group). (H) The concentrations of TGF-β1 detected by ELISA in supernatant from sorted macrophages of fibrotic kidneys (n = 5 for each group). (I) Representative flow cytometric images illustrating the expression of p-Smad2/3 + in CD45 + CD11b + F4/80 + live cells (left) and the percentage of p-Smad2/3 + cells in macrophages and its mean fluorescence intensity levels (2 parts in the right) in kidneys (n=6 for each group). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques: Knock-Out, Expressing, Fluorescence, Western Blot, Analysis, Control, Enzyme-linked Immunosorbent Assay, FACS, Real-time Polymerase Chain Reaction

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Inhibition of Dectin-1 attenuates chronic kidney disease (CKD) progression induced by unilateral ureteric obstruction (UUO) and ischemia-reperfusion (IR) in mice. (A-G) Laminarin was administered (300mg/kg/day) following sham or UUO surgery. (A) Experimental design of the treatment procedure. (B) Representative photomicrographs illustrating H&E and Masson's trichrome staining in kidney tissues. Scale bar, 50 μm. (C) Representative photomicrographs illustrating immunohistochemical staining for fibronectin, collagen1, vimentin, and α-SMA in kidney tissues (n=6 for each group). Scale bar, 50 μm. (D) Quantification of the positive staining area (%) in (C). (E) Representative western blot analysis of fibronectin, collagen1, vimentin, and α-SMA protein in the kidney tissues. GAPDH was used as loading control (n = 6 for each group). (F) Densitometric quantification of blots in (E). (G) Relative mRNA expression of Fn1 , Col1a1 , Vim , and Acta2 in kidney tissues (n = 6 for each group). (H-N) Laminarin was administered (300mg/kg/day) following sham or IR surgery. (H) Experimental design of the treatment procedure. (I) Representative photomicrographs illustrating H&E and Masson's trichrome staining in kidney tissues. Scale bar, 50 μm. (J) Representative photomicrographs illustrating immunohistochemical staining for fibronectin, collagen1, vimentin, and α-SMA in kidney tissues (n=6 for each group). Scale bar, 50 μm. (K) Quantification of the positive staining area (%) in (J). (L) Representative western blot analysis of fibronectin, collagen1, vimentin, and α-SMA protein in the kidney tissues. α-Tubulin was used as loading control (n = 6 for each group). (M) Densitometric quantification of blots in (L). (N) Relative mRNA expression of Fn1 , Col1a1 , Vim , and Acta2 in kidney tissues (n = 6 for each group). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques: Inhibition, Staining, Immunohistochemical staining, Western Blot, Analysis, Control, Expressing, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Dectin-1 is positively correlated with CCL2 and TGF-β1 in the kidneys of patients with chronic kidney disease (CKD). (A) Representative photomicrographs illustrating immunohistochemical staining for CCL2 and TGF-β1 in renal tissue of patients with IgA nephropathy (IgAN) (n = 42 for each group). Scale bar, 50 μm. (B) Quantification of the positive staining area (%) in (A). (C) The correlation of the number of Dectin-1 + cells with CCL2 and TGF-β1 expression in renal tissue of IgAN patients (n = 84). (D) The correlation of CLEC7A with CCL2 and TGFB1 in renal tissue of CKD patients in cohort 1 from GSE137570 (n = 24). (E) The correlation of CLEC7A with CCL2 and TGFB1 in renal tissue of CKD patients in cohort 2 from GSE137570 (n = 17). Data are presented as mean ± SD. **** P < 0.0001.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques: Immunohistochemical staining, Staining, Tissue, Expressing, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Correlation of the number of Dectin-1 + cells with major clinicopathological parameters in IgAN patients.

Article Snippet: The reagents used in this study, included the Dectin-1 antagonist laminarin (L9634, Sigma, USA), the Dectin-1 agonist depleted zymosan (d-zymosan, tlrl-zyd, InvivoGen, USA), the spleen tyrosine kinase (Syk) inhibitor R406 (S2194, Selleck, USA), the NF-κB inhibitor PDTC (P8765, Sigma, USA), the recombinant mouse transforming growth factor-β1 (TGF-β1, 7666-MB, R&D System, USA), mouse tumor necrosis factor-alpha (TNF-α) ELISA kits (430907, BioLegend, USA), mouse ELISA kits for C-C motif chemokine ligand 2 (CCL2, MJE00B, R&D Systems, USA), TGF-β1 (DB100C, R&D Systems, USA), interleukin (IL)-6 (M6000B, R&D Systems, USA), and IL-1β (MLB00C, R&D Systems, USA).

Techniques:

Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMP9 and BMP10 selectively activate SMAD1/5/8 signaling and induce proliferation in pulmonary artery endothelial cells but not pulmonary artery smooth muscle cells. ( A ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PAECs treated with the indicated TGF-β superfamily ligands (0.8 nM) or untreated control (UT); β-actin serves as a loading control. ( B ) PAEC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. ( C ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PASMCs treated with the indicated ligands (0.8 nM); β-actin serves as a loading control. ( D ) PASMC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. Data are shown as mean ± SD ( n = 3 replicate wells). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Control, BrdU Incorporation Assay

Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMPR2 dosage-dependent model for BMP9/10 signaling output in pulmonary artery endothelial cells. Schematic illustrating how BMPR2 abundance constrains BMP9/10 (ALK1-dependent) canonical signaling output and downstream cellular programs in PAECs. ( A ) BMPR2-sufficient (~100%) state: BMP9/10 predominantly signal through ALK1–BMPR2 complexes, generating pSMAD1/5/8 output consistent with a threshold-like requirement for proliferation; bimagrumab (BiMab) produces no effect detected under BMPR2-replete conditions. ( B ) BMPR2-limiting (~50%) state: Reduced BMPR2 attenuates BMP9/10-induced canonical output and is associated with reduced proliferation and increased caspase-3/7 activity consistent with stress/injury. Under BMPR2-limiting conditions, residual canonical output becomes bimagrumab-sensitive, consistent with context-dependent contribution of Activin type II receptors (predominantly ACVR2A in PAECs; see for BMP10 affinity comparisons) to the remaining pSMAD1/5/8 signal. A putative non-canonical stress-signaling arm is shown as a proposed intermediate. Solid arrows denote observed relationships; dashed arrows and dashed-outline boxes denote proposed steps. Node shading and output gauges depict relative canonical signaling output.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 1. Ntrk2 is up-regulated in mouse lungs after 24 hours of hypoxia exposure (pO2 0.01). Total RNA from mice lung homogenates was isolated (n 18 per group), direct labeled, and hybridized to 44K 60mer oligonucleotide microarrays (MWG). A: Volcano plot: log2-fold regulation compared to probability of regulation. Genes with log odds values 5 were considered to be regulated; black spots, exemplary genes. B: Functional annotation of regulated genes was performed according to Gene Ontology (GO). Ntrk2 (TrkB) belongs to MAPK signaling pathway. C: A time course of TrkB expression was performed using real-time PCR of lung homogenates harvested 1, 7, or 21 days after hypoxia exposure (n 7 to 9, each). D: Immunohistochemical staining of TrkB and its ligand, BDNF in mouse lung sections. The arrow indicates positive staining. E and F: Real-time PCR analysis of TrkB and BDNF expression in (E) isolated major pulmonary arteries (n 6), and (F) laser-microdissected intrapulmonary vessels (40 vessels per animal, n 4), from mice exposed to 21 days of hypoxia or normoxia. G: Expression levels of TrkB and BDNF in mouse PASMC as indicated by real-time PCR; lower CT values represent a more abundant transcript level. H: Immunofluorescence staining of TrkB and BDNF in mouse PASMC. The arrows indicate TrkB localization. Inset, negative control staining. *P 0.05.

Article Snippet: To investigate proliferation of unstimulated or BDNF-stimulated human PASMC, cells were permeabilized for 5 minutes with 0.25% Triton X-100 in Tris-buffered saline, incubated with a mouse anti-human Ki-67 antibody (Santa Cruz Biotechnology), followed by incubation with fluorescein isothiocyanate–labeled secondary antibody (Dianova, Hamburg, Germany).

Techniques: Isolation, Labeling, Functional Assay, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Immunofluorescence, Negative Control

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 3. Up-regulation of TrkB and BDNF in human lung tissue from IPAH. A: Colocalization of BDNF and TrkB with SMA as indicated by immunofluorescence staining in donor and IPAH patients. B: Real-time PCR analysis of TrkB and BDNF in laser-microdissected intrapulmonary vessels from donor and IPAH patients. Expression is given in comparison to samples from donor lungs (n 5 per group). C: Real-time PCR analysis of expression levels of TrkB and BDNF in human PASMC; lower CT values represent a more abundant transcript level. D: Immunofluorescence staining of TrkB and BDNF in human PASMC derived from donor and IPAH patients. E: Colocalization of BDNF and TrkB with SMA in PASMC as indicated by immunofluorescence staining, IgG, isotype control staining. *P 0.05.

Article Snippet: To investigate proliferation of unstimulated or BDNF-stimulated human PASMC, cells were permeabilized for 5 minutes with 0.25% Triton X-100 in Tris-buffered saline, incubated with a mouse anti-human Ki-67 antibody (Santa Cruz Biotechnology), followed by incubation with fluorescein isothiocyanate–labeled secondary antibody (Dianova, Hamburg, Germany).

Techniques: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Expressing, Comparison, Derivative Assay, Control

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 6. BDNF regulates and activates Egr-1. A: Induction of early growth-response factor 1 (Egr-1) protein in human PASMC as analyzed by Western blotting. B: BDNF-induced Egr-1 protein localization in PASMC as assessed by immunofluorescence analysis. C: Electrophoretic mobility shift assay for Egr-1 induction and DNA–protein interactions from nuclear extracts prepared from BDNF (10 ng/mL)-treated PASMC, incubated with radiolabeled oligonucleotide containing Egr-1 consensus sequence. Incubation with an excess (10-fold) of an unlabeled probe served as a control for specific binding. PDGF (10 ng/mL) treated cells served as a positive control. D: Egr-1 expression in lung parenchyma of donor and IPAH patients. An Egr-1/ mouse (KO-Egr-1) served as negative control. E: Real-time PCR analysis for Egr-1 expression in IPAH patient and donor laser-microdissected intrapulmonary vessels (n 5). n.s., not significant. *P 0.05.

Article Snippet: To investigate proliferation of unstimulated or BDNF-stimulated human PASMC, cells were permeabilized for 5 minutes with 0.25% Triton X-100 in Tris-buffered saline, incubated with a mouse anti-human Ki-67 antibody (Santa Cruz Biotechnology), followed by incubation with fluorescein isothiocyanate–labeled secondary antibody (Dianova, Hamburg, Germany).

Techniques: Western Blot, Immunofluorescence, Electrophoretic Mobility Shift Assay, Incubation, Sequencing, Control, Binding Assay, Positive Control, Expressing, Negative Control, Real-time Polymerase Chain Reaction

Journal: Journal of pharmacological sciences

Article Title: Protective effects of tadalafil on damaged podocytes in an adriamycin-induced nephrotic syndrome model.

doi: 10.1016/j.jphs.2022.03.003

Figure Lengend Snippet: Fig. 4. PDE5 expression in primary human podocyte cells. A) PCR and B) western blotting of PDE5 and Actb expression in positive control (human pulmonary artery smooth muscle cells) and primary human podocytes.

Article Snippet: Human pulmonary artery smooth muscle cells (PASMCs; ATCC, Manassas, VA, USA), passage 4, were used as positive controls for PDE5 in PCR and western blotting.

Techniques: Expressing, Western Blot, Positive Control

Journal: The American Journal of Pathology

Article Title: Exploratory Study of Prognostic Plasma Biomarkers in Patients with Pulmonary Arterial Hypertension

doi: 10.1016/j.ajpath.2025.04.018

Figure Lengend Snippet: Incremental predicted performance of identified biomarkers and expression in animal models. A: Performance of the logistic regression models with 2015 European Respiratory Society (ERS)/European Society of Cardiology (ESC) risk score, REVEAL 2.0 risk score, and the refined four-strata risk assessment method in patients with PAH for death and lung transplantation. Graph of C-statistics with 95% CI for each biomarker and their combinations. C-statistics were compared using the DeLong test with each reference. B: Heat map showing RNA-sequencing expression data of the indicated genes [in transcripts per million (TPM)] obtained from the Genotype-Tissue Expression (GTEx version 8) database ( https://gtexportal.org/home , last accessed August 13, 2024) in lung, heart, artery, kidney, liver, and adipose tissue. C: Western blot analyses and corresponding quantifications of WFDC2 (HE4) in right ventricles and dissected pulmonary arteries (PAs) from monocrotaline (MCT)–injected and Sugen/hypoxia (Su/Hx)–exposed rats. D: Western blot analyses and corresponding quantifications of WFDC2 (HE4) in kidney from MCT-injected and Su/Hx-exposed rats. E: Wfdc2 mRNA expression in right ventricle (RV), lung, and kidney of control (CTRL) and MCT-injected or Su/Hx-exposed rats. F: Representative fluorescent images of 5-ethynyl-2′-deoxyuridine (EdU)–labeled control pulmonary artery smooth muscle cells (PASMCs) and control cardiac fibroblasts (CFs) exposed or not to recombinant human WFDC2 (rhWFDC2; 40 nmol/L) for 24 and 48 hours, respectively. Ar row s indicate EdU-positive cells. The corresponding quantifications are shown. Scatter dot plots show individual values. Protein expression was normalized to amido black (AB). Statistical analyses were performed using t -test. Data are given as means ± SEM ( A and E ). n = 6 to 16 ( E ); n = 3 control PASMCs ( F ); n = 4 control CFs ( F ). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001. Scale bars = 100 μm ( F ). Veh, vehicle.

Article Snippet: Control pulmonary artery smooth muscle cells (PASMCs; n = 5 cell lines) were either purchased from Cell Applications (San Diego, CA) or isolated from patients without PAH.

Techniques: Expressing, Transplantation Assay, Biomarker Discovery, RNA Sequencing, Western Blot, Injection, Control, Labeling, Recombinant