Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease

doi: 10.1161/JAHA.113.000082

Figure Lengend Snippet: Viral RNA copy numbers were determined by RT‐qPCR. (A) Viral RNA copy numbers were compared among the Stage groups. (B) Single‐step growth curves of PAD‐HEV were examined at the various time points in the primary HSkm cells. Viral RNA copy numbers were determined using in vitro transcripts (see more detail in Supplemental Materials). PAD‐HEV1 (Stage III), PAD‐HEV2 (Stage II), PAD‐HEV3 and 4 (Stage IV). RT‐qPCR indicates reverse‐transcription real‐time quantitative polymerase chain reaction; PAD, peripheral arterial disease; HEV, human enterovirus; HSkm, primary human skeletal muscle; CVB1, coxsackievirus B1.

Article Snippet: HeLa cells (ATCC) were used for HEV propagation, HEV detection, and infectivity experiments., – Primary human skeletal muscle (HSkm) cells, cultured in MyoTonic Growth Medium (Cook MyoSite), also were used to study virus infectivity.

Techniques: Quantitative RT-PCR, In Vitro, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Haplodeficiency of Ataxia Telangiectasia Mutated Accelerates Heart Failure After Myocardial Infarction

doi: 10.1161/JAHA.117.006349

Figure Lengend Snippet: Senescence is involved in post–myocardial infarction ( MI) cardiac remodeling in mouse heart. A, Real‐time polymerase chain reaction (PCR) analysis of ataxia telangiectasia mutated (ATM), p53, and p21 mRNA levels at days 0, 3, and 7 after MI . B, Real‐time PCR analysis of ATM , p53, and p21 mRNA levels in sham mouse and different regions (remote area, border area, and infarct area) at day 7 post MI. n=4 in each group. * P <0.05 vs the day 0 or sham group. Western blot analysis and bar graph of (C) p21 expression in wild‐type mouse hearts at indicated times after sham or MI surgery, and (D) p21 expression in different regions of wild‐type mouse hearts 7 days after sham or MI surgery. E, Representative images and (F) statistical analysis of senescence‐associated β‐galactosidase (SA‐β‐gal) staining in mouse hearts at day 7 after sham or MI surgery. n=4 in each group. * P <0.05 vs the sham group, ** P <0.01 vs the sham group. G, Representative images of costaining of SA ‐β‐gal and α‐smooth muscle actin (α‐ SMA )/α‐actinin staining in heart section at day 7 after MI surgery. Bar=50 μm.

Article Snippet: Briefly, heart sections (5 μm) from paraffin‐embedded tissues were deparaffinized and incubated with anti‐mouse monoclonal antibodies against α‐smooth muscle actin (α‐SMA) (1:200 dilution, Santa Cruz Biotechnology) and CD31 (1:200 dilution, Cell Signaling Technology) at 4°C overnight.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Haplodeficiency of Ataxia Telangiectasia Mutated Accelerates Heart Failure After Myocardial Infarction

doi: 10.1161/JAHA.117.006349

Figure Lengend Snippet: Ataxia telangiectasia mutated (ATM) haplodeficiency increases myofibroblast accumulation and decreases fibroblast senescence both in vivo and in vitro. A, Quantitative real‐time polymerase chain reaction (PCR) was performed to detect the mRNA level of α‐smooth muscle actin (α‐ SMA ), collagen I, and collagen III in the hearts of ATM +/+ and ATM +/− mice at the indicated time points post‐myocardial infarction (MI). n=4 in each group. * P <0.05, # P <0.01 vs the corresponding sham group; † P <0.05 vs the control MI group. B, Western blot analysis of α‐ SMA levels in the hearts of ATM +/+ and ATM +/− mice at the indicated time points post MI . C, Representative collagen I–stained sections of infarct, border, and remote size 7 days after MI in ATM +/+ and ATM +/− mice. D, Western blot analysis of p21 levels in the hearts of ATM +/+ and ATM +/− mice at the indicated time points post MI . E, Representative images and statistical analysis of senescence‐associated β‐galactosidase (SA‐β‐gal) staining in cardiac sections from ATM +/+ and ATM +/− mice after MI . F, Representative images of costaining of p19 or p‐p53 with α‐ SMA in ATM +/+ and ATM +/− mice after MI . G, Representative images and statistical analysis of SA ‐β‐gal staining in isolated cardiac fibroblasts after hypoxia/oxygenation treatment and (H) cardiac fibroblasts from ATM +/+ and ATM +/− mice after MI surgery. n=4 in each group. * P <0.05 vs the corresponding ATM +/− group. Bar=50 μm.

Article Snippet: Briefly, heart sections (5 μm) from paraffin‐embedded tissues were deparaffinized and incubated with anti‐mouse monoclonal antibodies against α‐smooth muscle actin (α‐SMA) (1:200 dilution, Santa Cruz Biotechnology) and CD31 (1:200 dilution, Cell Signaling Technology) at 4°C overnight.

Techniques: In Vivo, In Vitro, Real-time Polymerase Chain Reaction, Control, Western Blot, Staining, Isolation

Journal:

Article Title: Acute Humanin Therapy Attenuates Myocardial Ischemia and Reperfusion Injury in Mice

doi: 10.1161/ATVBAHA.110.205997

Figure Lengend Snippet: HNG improves cardiomyocyte survival in vitro and decreases apoptosis in response to Daunorubicin. Effect of HNG on A) Cardiomyocyte survival as assessed by cell viability assay, * p < 0.05 Dauno compared to Dauno+HNG, and B) early markers of apoptosis as assessed by CaspASE FITC -VAD-FMK in situ apoptosis assay.

Article Snippet: Effect of HNG on cardiomyocyte survival and apoptosis in vitro Rat heart myocardium cell line H9c2 (2-1) (cat# CRL-1446) was purchased from ATCC.

Techniques: In Vitro, Viability Assay, In Situ, Apoptosis Assay

Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: Mexo-P regulates vascular calcification in a let-7b-5p-dependent way. ( A ) Volcano plots show the result of miRNA sequencing between Mexo- and Mexo-P-treated MOVAS. ( B ) qRT-PCR validation of potential target miRNAs between Mexo- and Mexo-P-treated HASMCs (unpaired t -test, n = 5). ( C ) Expression levels of let-7b-5p in the artery tissue of mildly, moderately, or severely calcified CKD patients (unpaired t -test, n > 3). ARS staining ( D ) and calcium contents (( E ), one-way ANOVA, n = 4) of isolated mouse thoracic aortas, cultured in a calcifying medium (3 mM Pi) for 7 days, pretreated with let-7b-5p agomir or antagomir for 6 h. Representative images of ARS staining ( F ) and ARS quantification ( G ) of MOVAS treated with let-7b-5p-mimic or control (mimic-nc) and cultured in the calcifying medium for 7 days (Mann–Whitney test, n = 4). Representative images of ARS staining ( H ) and ARS quantification ( I ) of MOVAS treated with let-7b-5p-inhibitor or control (inhibitor-nc) and cultured in the calcifying medium for 7 days (unpaired t -test, n = 4). Representative images of ARS staining ( J ) and ARS quantification ( K ) of HASMCs treated with let-7b-5p-mimic or control and cultured in the calcifying medium for 7 days (unpaired t -test, n = 4). Representative images of ARS staining ( L ) and ARS quantification ( M ) of MOVAS treated with let-7b-5p-inhibitor or control and cultured in the calcifying medium for 7 days (unpaired t -test, n = 4). Calcium content levels of calcifying-medium-cultured VSMCs (MOVAS, ( N ), HASMC, ( O )), stimulated with Mexo or Mexo-P in the presence or absence of let-7b-5p-mimic (one-way ANOVA, n = 4). * p < 0.05, *** p < 0.001, **** p < 0.0001, n.s. not significant. Scale bar, 40 μM.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Sequencing, Quantitative RT-PCR, Biomarker Discovery, Expressing, Staining, Isolation, Cell Culture, Control, MANN-WHITNEY

Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: Let-7b-5p mitigates vascular calcification by regulating TGFBR1. ( A ) Volcano plots showing differentially regulated genes (DEGs) in RNA-seq of Mexo- and Mexo-P-treated MOVAS. ( B ) Venn diagram showing the intersection of miRBD-predicted potential let-7b-5p targets and DEGs. ( C ) mRNA levels of 10 potential target genes were detected by qRT-PCR in let-7b-5p-mimic- or mimic-nc-treated HASMCs (unpaired t -test, n > 3). ( D ) Transcription levels of four significantly regulated genes in ( C ) were detected in Mexo- or Mexo-P-treated HASMC (unpaired t -test, n > 3). ( E ) Representative images of ARS staining (bottom) and ARS quantification (top) of HASMCs, in which TGFBR1 and let-7b-5p levels were manipulated with TGFBR1 overexpression plasmid (TGFBR1-OE) or let-7b-5p-mimic treatment in calcifying medium (one-way ANOVA, n = 4). ( F ) Representative images of ARS staining (bottom) and ARS quantification (top) of HASMCs, in which TGFBR1 and let-7b-5p levels were manipulated with TGFBR1-si or let-7b-5p-mimic treatment in calcifying medium (one-way ANOVA, n = 4). Calcium content of HASMCs with altered TGFBR1 levels with TGFBR1-OE ( G ) or TGFBR1-si ( H ), with or without the treatment of let-7b-5p-mimic or let-7b-5p-inhibitor in calcifying medium (one-way ANOVA, n = 4). ( I ) Tergetscan database predicted binding sequences of let-7b-5p on TGFBR1-3′UTR. ( J ) Wildtype (wt) and two mutations (mut1 and mut2) of TGFBR1-3′UTR were inserted into luciferase vectors. Luciferase activity assay of HASMCs co-transfected with vectors carrying wt or mutated TGFBR1-3′UTR (mut1, ( K ), mut2, ( L )), renilla vectors, mimic-nc, or let-7b-5p-mimic for 6 h, medium change, and cultured for another 48 h before assay (two-way ANOVA, n > 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: RNA Sequencing, Quantitative RT-PCR, Staining, Over Expression, Plasmid Preparation, Binding Assay, Luciferase, Activity Assay, Transfection, Cell Culture

Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: Manipulating TGFBR1 levels regulates vascular calcification in CKD. Transcription levels of RUNX2, ALPL, and SOX9 in MOVAS ( A ) and HASMCs ( C ) stimulated with 10 ng/mL TGFβ1 for 24 h after being transfected with TGFBR1 siRNA for 6 h (one-way ANOVA, n > 3). mRNA levels of RUNX2, ALPL, and SOX9 in MOVAS ( B ) and HASMCs ( D ) stimulated with 10 ng/mL TGFβ1 for 24 h after being transfected with TGFBR1 overexpression plasmid (TGFBR1-OE) for 48 h (one-way ANOVA, n > 3). Representative WB images ( E ) and semi-quantification ( F ) showing RUNX2 levels in aortas from CKD mice injected with either Tgfbr1-adv or control (GFP-adv, unpaired t -test, n = 3). Representative images of ARS staining ( G ) and calcium content ( H ) of aortas from CKD mice injected with either Tgfbr1-adv or control (unpaired t -test, n > 3). ( I ) HASMCs were treated with SB525334 (1 μM), RepSOX (25 μM), and SB431542 (1 μM) for 6 h, after being transfected with SMAD3-luciferase plasmids for 24 h, and luciferase activity was detected (unpaired t -test, n = 3). ( J ) SMAD3-luciferase plasmid-transfected HASMCs were stimulated with 10 ng/mL TGFβ1 and different doses of SB525334 for 6 h, and luciferase activity was detected (one-way ANOVA, n > 3). ( K ) HASMCs were stimulated with 10 ng/mL TGFβ1 plus 1 μM SB525334 for 0, 3, 6, 12, 24, or 48 h, and RUNX2 mRNA levels were detected by qRT-PCR (one-way ANOVA, n = 5). In vitro calcification model showing the SB525334 (1 μM) inhibition of 10 ng/mL TGFβ1-induced HASMC calcification (( L ), ARS staining, ( M ), calcium content, one-way ANOVA, n = 6). Mice were orally administrated with 30 mg/kg SB525334 (n = 5) or vehicle (n = 5) daily; simultaneously with CKD modeling, aortas were collected for ARS staining ( N ), calcium content assay (( O ), unpaired t -test, n > 3), and Runx2, Alpl, and Sox9 mRNA levels detection (( P ), unpaired t -test, n > 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant. Scale bar, 40 μM.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Transfection, Over Expression, Plasmid Preparation, Injection, Control, Staining, Luciferase, Activity Assay, Quantitative RT-PCR, In Vitro, Inhibition

Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: TGFBR1 promotes vascular calcification through the SMAD3/RUNX2 pathway. qRT-PCR results showing that SIS-3 reversed TGFβ1-induced upregulation of RUNX2 levels in HASMCs ( A ) and MOVAS (( B ), one-way ANOVA, n = 5). Western blot images showing that different doses of SMAD3 inhibitor SIS-3 alleviated TGFβ1-induced RUNX2 level increase and SMAD2/3 phosphorylation in HAMSCs ( C ) and MOVAS ( D ). ( E ) The JASPAR database predicted the binding site of SMAD3 on RUNX2 promoter, and the mutation was generated. ( F ) HASMCs were transfected with Renilla plasmid, wildtype RUNX2-promoter luciferase plasmid, and different doses of SMAD3-OE plasmid for 24 h, and luciferase activity was detected (one-way ANOVA, n = 5). ( G ) HASMC was transfected with Renilla plasmid, SMAD3-OE plasmid (3 ng per well of 12-well plate), and wildtype (wt) or mutated (mut) RUNX2-promoter luciferase plasmid, and luciferase activity was detected (Mann–Whitney test, n = 6). ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Western Blot, Phospho-proteomics, Binding Assay, Mutagenesis, Generated, Transfection, Plasmid Preparation, Luciferase, Activity Assay, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are localized and secreted by human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A Human lung tissue from control subjects, idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF was immune-stained with IL-11, IL-11Rα and αSMA and with secondary fluorescence antibodies. Representative images are showed. White colour represents co-localization of both antibodies. Yellow arrows indicate endothelial cells. B HPAECs and C HPASMCs were isolated from pulmonary arteries of control subjects, IPF and PH associated to IPF patients and cultured until passage 1. Cell culture supernatants were collected to measure IL-11 by ELISA. Data are presented as scatter dot blot with median and interquartile range values of n = 6 patients in each group. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Staining, Fluorescence, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are increased in whole lung homogenates, isolated pulmonary arteries and serum of patients with idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF. The protein expression of IL-11 and IL-11Rα in A , B isolated pulmonary arteries (70–500 µm of internal diameter), C , D serum, and E , F lung tissue homogenates. Protein expression was measured using ELISA kits. H CD31 protein expression was measured in isolated pulmonary arteries as endothelial cells marker by ELISA. H , I Human lung tissue from control subjects, IPF and PH associated to IPF was immune-stained with IL-11, IL-11Rα and alpha smooth muscle actin (αSMA) antibodies. Representative images are showed from non-fibrotic lung areas and fibrotic areas. J Vascular wall thickening was quantified in a total of 20–30 pulmonary arteries per patient. K , L Immunohistochemical score quantification of IL-11 and IL-11Rα in a total of 20–30 pulmonary arteries per patient. Scale bar: 100 µm. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison. M Spearman ρ correlation of IL-11 expression in isolated pulmonary arteries from PH + IPF and mean pulmonary artery pressure (mPAP). N indicates the number of patients in each graph

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Marker, Staining, Immunohistochemical staining, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: SiRNA-IL-11 transiently transfection attenuates bleomycin-induced lung fibrosis and pulmonary hypertension in transgenic Tie2-GFP mice. Wild-type (WT) siRNA(−) Tie2-GFP mice and IL-11-KO siRNA-IL-11 Tie2-GFP mice received a single intratracheal dose of bleomycin (1.5 U/kg) on day 1 ( n = 11) during 14 days. siRNA-IL-11 was administered intravenously and intranasally three times a week from day 1 to day 14. At day 14 the following parameters were measured. A Masson’s trichrome histological images are showed. Scale bar: 100 µm. B Ashcroft score lung fibrotic index, C hydroxyproline amount in lung tissue D right ventricular systolic pressure (RVSP) mmHg, E right ventricular (RV) hypertrophy measured by the ratio of RV/left ventricular (LV) + septo in mg/mg, F pulmonary artery remodeling and G inflammatory cells in bronchoalveolar lavage fluid (BALF) were measured. H Immunohistochemical analysis of αSMA, IL-11 and IL-11Rα. Scale bar: 50 µm. Black arrows show pulmonary arteries. I Co-immunofluorescence of αSMA/Tie2-GFP. Scale bar: 25 µm. White arrows indicates co-localizations. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Transfection, Transgenic Assay, Immunohistochemical staining, Immunofluorescence, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and soluble IL-11Rα induce human pulmonary artery endothelial cell (HPAEC) to mesenchymal transition (EnMT) and human pulmonary artery smooth muscle cell (HPASMC) to myofibroblast-like transition. A HPAEC and B HPASMC were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or their combination during 48 h replacing culture medium and stimulus each 24 h. Experiments were done between passages 2–3. Gene mRNA transcripts of different genes measured by quantitative PCR (qPCR) as 2 −ΔCt . Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin for protein. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 4 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα activates intracellular signal. A Human pulmonary artery endothelial cells (HPAEC) and B human pulmonary artery smooth muscle cells (HPASMC) were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination during 30 min. Experiments were done between passages 2–3. Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin or non-phosphorylated protein as indicate. Representative blots are sowed. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα promotes time-dependent proliferation and senescence in human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A HPAECs or B HPASMCs were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination at indicated times. Experiments were done between passages 2–3. Cell proliferation was measured by the BrDU kit at 24 h, 48 h, 72 h and 96 h. Cell senescence was measured after 72 h of cell stimulation using β-galactosidase histology and P21 expression. Results were expressed as % senescence (β-galactosidase blue positive cells) relative to the total number of cells in each field. P21 expression was measured by quantitative PCR (qPCR) as 2 −ΔCt and western blot. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3–4 control subjects performed in triplicate). P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Cell Stimulation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Dot Blot

Journal: Journal of Clinical Investigation

Article Title: Lymph node fibroblastic reticular cells deposit fibrosis-associated collagen following organ transplantation

doi: 10.1172/jci136618

Figure Lengend Snippet: Figure 2. Repetitive skin trans- plantation induces fibrosis in DLNs. (A) Collagen I fibers (red) in the DLNs (axillary LNs, DLNRep) of mice following repetitive skin transplantation in comparison with the axillary LNs of age- matched naive mice and semi- quantitative analysis. Scale bar: 1500 μm. n = 5. (B) Costaining of Meca-79+ HEVs (green) and Lyve-1+ lymphatic vessels (green) with collagen I fibers (red) and fibronectin fibers (red) in DLNRep and age-matched naive LNs. Scale bars: 100 μm. (C) Masson’s trichrome stain of fibrosis in DLNRep and age-matched naive LNs. Scale bars: 50 μm. (D) Flu- orescence micrographs showing expression of the myofibroblast marker α-SMA by PDPN+ FRCs in DLNRep and naive LNs. Scale bars: 100 μm. (E) Gene expression lev- els of fibrosis markers and TGF-β signaling molecules in the DLNRep and LNs of naive mice. n = 4. The percentage of the areas stained positive in the fluorescence micrographs was assessed in 3–6 random microscopic fields for each mouse. Data are presented as the mean ± SD. *P < 0.05 and **P < 0.01, by Student’s t test.

Article Snippet: We used the following primary antibodies: mouse fibronectin (Abcam, ab2413; 1:1000); rabbit anti–mouse αSMA (Cell Signaling Technolo gy, 19245, 1:1000); rabbit anti–mouse p21Waf1/Cip1 (Cell Signaling Tech nology, 2947, 1:1000); rabbit anti–mouse CDKN2A/p16INK4a (Abcam, ab2D9A12, 1:1000); and mouse anti–mouse GADPH (Santa Cruz Bio technology, sc32233, 1:100).

Techniques: Transplantation Assay, Comparison, Staining, Expressing, Marker, Gene Expression, Fluorescence

Journal: Journal of Clinical Investigation

Article Title: Lymph node fibroblastic reticular cells deposit fibrosis-associated collagen following organ transplantation

doi: 10.1172/jci136618

Figure Lengend Snippet: Figure 3. Senescent FRCs play a crucial role in the accumulation of ECM following transplantation. (A) Representative images of β-gal (senescence marker) staining in DLNRep and a LN of an age-matched naive mouse. Scale bars: 50 μm. (B) Costaining of PDPN+ FRCs (red) and the senescence marker p21Waf1/Cip1 (p21) in sections of DLNRep and an age-matched naive LN. Scale bars: 50 μm. (C) Expression levels of senescence genes in DLNRep and an age-matched naive LN. n = 4 (each dot represents 1 sample). (D) Staining with β-gal and (E) costaining with PDPN (red) for p21, collagen I, and α-SMA in FRCs from a naive LN and DLNRep. Scale bars: 50 μm. (F) Gene expression levels of Il6, Col1a1, and Acta2 in FRCs following in vitro treatment with the senescence inducer etoposide (Eto). (G) Schematic of the generation of CCL19Cre iDTR mice. (H) Collagen I+ fibers (red) in the DLNs of CCL19Cre iDTR allogeneic skin transplant recipient mice with or without DT treatment. Scale bars: 50 μm. (I) Gene expression levels of Cdkn2a, Cdkn1a, Acta2, and Col1a1 in DLNs from CCL19Cre iDTR allogeneic skin transplant recipient mice with or without DT treatment (n = 6). (J) Schematic of the generation of CCL19Cre p16fl/fl mice with FRC conditional knockout of p16. (K) Collagen I fibers (red) in DLNs of C57BL/6J and CCL19Cre p16fl/fl recipient mice following allogeneic repetitive skin transplantation. Scale bars: 50 μm. (L) Gene expression levels of Col1a1, Fn1, and Acta2 in DLNs (n = 6). The percentage of areas stained positive in the fluorescence micrographs was assessed in 3–6 random microscopic fields for each mouse. Data are presented as the mean ± SD. *P < 0.05 and **P < 0.01, by Student’s t test.

Article Snippet: We used the following primary antibodies: mouse fibronectin (Abcam, ab2413; 1:1000); rabbit anti–mouse αSMA (Cell Signaling Technolo gy, 19245, 1:1000); rabbit anti–mouse p21Waf1/Cip1 (Cell Signaling Tech nology, 2947, 1:1000); rabbit anti–mouse CDKN2A/p16INK4a (Abcam, ab2D9A12, 1:1000); and mouse anti–mouse GADPH (Santa Cruz Bio technology, sc32233, 1:100).

Techniques: Transplantation Assay, Marker, Staining, Expressing, Gene Expression, In Vitro, Knock-Out, Fluorescence

Journal: Journal of Clinical Investigation

Article Title: Lymph node fibroblastic reticular cells deposit fibrosis-associated collagen following organ transplantation

doi: 10.1172/jci136618

Figure Lengend Snippet: Figure 4. LIGHT increases ECM accumulation in DLNs by binding to HVEM in FRCs following transplantation. (A) IF staining for HVEM (green) expres- sion in PDPN+ FRCs (red) in vitro. Scale bar: 10 μm. (B) Flow cytometric analysis of HVEM expression by FRCs through gating on the CD45–CD31–PDPN+ cell population from LNs. (C) Staining and semiquantitative analysis of collagen I fibers (red) in DLNRep (staining was normalized to DAPI). Scale bars: 10 μm. n = 4. (D) Gene expression levels of senescence and fibrosis genes in FRCs from WT and HVEM-KO mice treated with LIGHT (25 ng/mL). n = 6. (E) Western blot and quantification of protein levels of fibronectin, α-SMA, p21, and p16 in cultured WT FRCs after treatment with LIGHT (25 ng/mL). (F) IF staining of cultured WT FRCs with collagen I (green) and PDPN (red) after LIGHT (25 ng/mL) treatment compared with no treatment. Scale bars: 10 μm. (G) Measure- ment of protein levels of fibronectin and p16 in cultured HVEM-KO FRCs after LIGHT stimulation. (H) IF staining for LIGHT expression in mast cells. Scale bar:10 μm. The percentage of areas stained positive in the fluorescence micrographs was assessed in 3–6 random microscopic fields for each mouse. Data are presented as the mean ± SD. *P < 0.05 and **P < 0.01, by Student’s t test.

Article Snippet: We used the following primary antibodies: mouse fibronectin (Abcam, ab2413; 1:1000); rabbit anti–mouse αSMA (Cell Signaling Technolo gy, 19245, 1:1000); rabbit anti–mouse p21Waf1/Cip1 (Cell Signaling Tech nology, 2947, 1:1000); rabbit anti–mouse CDKN2A/p16INK4a (Abcam, ab2D9A12, 1:1000); and mouse anti–mouse GADPH (Santa Cruz Bio technology, sc32233, 1:100).

Techniques: Binding Assay, Transplantation Assay, Staining, In Vitro, Expressing, Gene Expression, Western Blot, Cell Culture, Fluorescence