Journal: ESC Heart Failure

Article Title: Transcriptomes of human mesenchymal cells isolated from the right ventricle and epicardial fat differ strikingly both directly after isolation and long‐term culture

doi: 10.1002/ehf2.12397

Figure Lengend Snippet: Immunophenotyping of freshly isolated cells from the right ventricle and epicardial fat. Representative gating strategy utilized to sort CD9031 − CD45 − CD90 + CD34 + CD146 − cells from the right ventricle and epicardial fat.

Article Snippet: Subsequently, PBS was added to 50 mL, and cells were centrifuged (300 g , 10 min, RT), resuspended in 100 μL PBS with 1% FBS, and incubated with the following set of mouse anti‐human monoclonal antibodies (20 min, 4°C): anti‐CD90‐PE (20 μL, Miltenyi Biotec), anti‐CD34‐APC (20 μL, Miltenyi Biotec), anti‐CD31‐APC Cy7 (5 μL, BD Biosciences), anti‐CD45‐PE Cy7 (5 μL, BD Biosciences), and anti‐CD146‐FITC (5 μL, BD Biosciences).

Techniques: Isolation

Journal: ESC Heart Failure

Article Title: Transcriptomes of human mesenchymal cells isolated from the right ventricle and epicardial fat differ strikingly both directly after isolation and long‐term culture

doi: 10.1002/ehf2.12397

Figure Lengend Snippet: RNA‐seq analysis of freshly isolated cells. (A) A number of transcripts detected in samples isolated from the right ventricle (HEARTS) and epicardial fat (FAT). (B) Principal component analysis (PCA) of transcripts detected in cells isolated from both tissues. HEART: CD31 − CD45 − CD90 + CD34 + CD146 − cells isolated from the right ventricle (1, 2, 3, 4, 5—patient ID). FAT: CD31 − CD45 − CD90 + CD34 + CD146 − cells isolated from epicardial fat (1, 2, 4, 5—patient ID). (C) Hierarchical clustering based on differentially expressed transcripts detected in cells from both tissues.

Article Snippet: Subsequently, PBS was added to 50 mL, and cells were centrifuged (300 g , 10 min, RT), resuspended in 100 μL PBS with 1% FBS, and incubated with the following set of mouse anti‐human monoclonal antibodies (20 min, 4°C): anti‐CD90‐PE (20 μL, Miltenyi Biotec), anti‐CD34‐APC (20 μL, Miltenyi Biotec), anti‐CD31‐APC Cy7 (5 μL, BD Biosciences), anti‐CD45‐PE Cy7 (5 μL, BD Biosciences), and anti‐CD146‐FITC (5 μL, BD Biosciences).

Techniques: RNA Sequencing Assay, Isolation

Journal: ESC Heart Failure

Article Title: Transcriptomes of human mesenchymal cells isolated from the right ventricle and epicardial fat differ strikingly both directly after isolation and long‐term culture

doi: 10.1002/ehf2.12397

Figure Lengend Snippet: RNA‐seq analysis of in vitro expanded cells (passage 6). (A) Number of transcripts detected in samples isolated from the right ventricle (HEARTS) and epicardial fat (FAT). (B) Principal component analysis (PCA) of transcripts detected in cells isolated from both tissues. HEART: CD31 − CD45 − CD90 + CD34 + CD146 − cells isolated from right ventricle (1, 2, 3, 4, 5—patient ID). FAT: CD31 − CD45 − CD90 + CD34 + CD146 − cells isolated from epicardial fat (2, 3, 4, 5—patient ID). (C) Hierarchical clustering based on differentially expressed transcripts detected in cells from both tissues. (D) Hierarchical clustering based on 40 most differentially expressed transcripts detected in cells from both tissues.

Article Snippet: Subsequently, PBS was added to 50 mL, and cells were centrifuged (300 g , 10 min, RT), resuspended in 100 μL PBS with 1% FBS, and incubated with the following set of mouse anti‐human monoclonal antibodies (20 min, 4°C): anti‐CD90‐PE (20 μL, Miltenyi Biotec), anti‐CD34‐APC (20 μL, Miltenyi Biotec), anti‐CD31‐APC Cy7 (5 μL, BD Biosciences), anti‐CD45‐PE Cy7 (5 μL, BD Biosciences), and anti‐CD146‐FITC (5 μL, BD Biosciences).

Techniques: RNA Sequencing Assay, In Vitro, Isolation

Journal: ESC Heart Failure

Article Title: Transcriptomes of human mesenchymal cells isolated from the right ventricle and epicardial fat differ strikingly both directly after isolation and long‐term culture

doi: 10.1002/ehf2.12397

Figure Lengend Snippet: Comparison of freshly isolated and in vitro expanded epicardial fat‐derived cells. (A) Principal component analysis (PCA) of transcripts detected in freshly isolated and in vitro expanded CD31 − CD45 − CD90 + CD34 + CD146 − cells isolated from epicardial fat (FAT). Before: freshly isolated cells; after: in vitro expanded cells (1, 2, 3, 4, 5—patient ID). (B) Gene ontology terms overrepresented among the genes differentiating (adj. P ‐value <0.05) freshly isolated and in vitro expanded cells down‐regulated after cell culture. (C) Gene ontology terms overrepresented among the genes differentiating (adj. P ‐value <0.05) freshly isolated and in vitro expanded cells up‐regulated after cell culture.

Article Snippet: Subsequently, PBS was added to 50 mL, and cells were centrifuged (300 g , 10 min, RT), resuspended in 100 μL PBS with 1% FBS, and incubated with the following set of mouse anti‐human monoclonal antibodies (20 min, 4°C): anti‐CD90‐PE (20 μL, Miltenyi Biotec), anti‐CD34‐APC (20 μL, Miltenyi Biotec), anti‐CD31‐APC Cy7 (5 μL, BD Biosciences), anti‐CD45‐PE Cy7 (5 μL, BD Biosciences), and anti‐CD146‐FITC (5 μL, BD Biosciences).

Techniques: Isolation, In Vitro, Derivative Assay, Cell Culture

Journal: ESC Heart Failure

Article Title: Transcriptomes of human mesenchymal cells isolated from the right ventricle and epicardial fat differ strikingly both directly after isolation and long‐term culture

doi: 10.1002/ehf2.12397

Figure Lengend Snippet: Comparison of freshly isolated and in vitro expanded cells. (A) Principal component analysis (PCA) of transcripts detected in freshly isolated and in vitro expanded CD31 − CD45 − CD90 + CD34 + CD146 − cells isolated from the right ventricle (HEART). Before: freshly isolated cells; after: in vitro expanded cells (1, 2, 3, 4, 5—patient ID). (B) Gene ontology terms overrepresented among the genes differentiating (adj. P ‐value <0.05) freshly isolated and in vitro expanded cells down‐regulated after cell culture. (C) Gene ontology terms overrepresented among the genes differentiating (adj. P ‐value <0.05) freshly isolated and in vitro expanded cells up‐regulated after cell culture.

Article Snippet: Subsequently, PBS was added to 50 mL, and cells were centrifuged (300 g , 10 min, RT), resuspended in 100 μL PBS with 1% FBS, and incubated with the following set of mouse anti‐human monoclonal antibodies (20 min, 4°C): anti‐CD90‐PE (20 μL, Miltenyi Biotec), anti‐CD34‐APC (20 μL, Miltenyi Biotec), anti‐CD31‐APC Cy7 (5 μL, BD Biosciences), anti‐CD45‐PE Cy7 (5 μL, BD Biosciences), and anti‐CD146‐FITC (5 μL, BD Biosciences).

Techniques: Isolation, In Vitro, Cell Culture

Journal: Oncotarget

Article Title: Capsaicin triggers autophagic cell survival which drives epithelial mesenchymal transition and chemoresistance in bladder cancer cells in an Hedgehog-dependent manner

doi: 10.18632/oncotarget.10326

Figure Lengend Snippet: A. Cell death was evaluated in BC cells, untreated or treated for different times with CPS (300 μM) alone or in combination with BAF (25 nM) by FACS analysis. Data shown are the mean ± SD of three independent experiments. *p<0.01 vs untreated; #p<0.01 CPS plus BAF vs CPS- or BAF-treated cells. B. Cell death type was determined by Annexin V/PI staining and cytofluorimetric analysis in BC cells after 72 h of treatment with CPS (300 μM) alone or in combination with BAF (25 nM). Data are representative of one out of three separate experiments. Numbers represent the percentage of positive cells. C. Lysates from siGLO and siBeclin 1 BC cells were separated on 8% SDS-PAGE and probed with anti-Beclin 1 Ab. Cropped blots are representative of one of three separate experiments. Bars represent the densitometric analysis, shown as the mean ± SD of three different experiments, evaluated using siGLO cells as calibrator. *p<0.01 siBeclin 1 vs siGLO cells. D. Lysates from siGLO and siBeclin 1 BC cells treated for 72 h with CPS (300 μM) were separated on 14% SDS-PAGE and probed with anti-LC3 Ab. Cropped blots are representative of one of three separate experiments. Bars represent the densitometric analysis, shown as the mean ± SD of three different experiments, evaluated using siGLO cells as calibrator. *p<0.01 CPS-treated siBeclin 1 vs CPS-treated siGLO cells. E. Cell viability was assessed by MTT assay in siGLO and siBeclin 1 BC cells treated for 72 h with CPS (300 μM). Data shown are the mean ± SD of three independent experiments. *p<0.01 siBeclin 1 vs siGLO CPS-treated BC cells.

Article Snippet: Annexin V-FITC (5 μl/ml) was purchased from Enzo Life Sciences (NY, USA).

Techniques: Staining, SDS Page, MTT Assay

Journal:

Article Title: Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA

doi: 10.1261/rna.1279909

Figure Lengend Snippet: Detection of individual GAPDH transcripts in situ. Reactions in situ were carried out under the conditions described in Materials and Methods. The phosphorylated oligonucleotides PP5 (specific for GAPDH) (A,B,E,F) were used as specific tools for GAPDH transcript detection in situ. Nonspecific phosphorylated oligonucleotides PP6 (differing from PP5 in few nucleotides in ligation junction region) (C,D) were used as controls of reaction specificity. The RCA reaction products were hybridized with FITC containing oligonucleotides and slides were analyzed using confocal microscope with 40× (A,C) and 63× (B,D–F) objectives. For best resolution of individual cells fivefold stronger magnification was used (E,F). The images are presented as an overlay of DAPI and FITC fluorescence projections and single bright field image. The green FITC label spots in cell cytoplasm surrounding blue-colored nuclei indicate the locations of specifically labeled RCA products. The specific RCA product hybridized with FITC-labeled oligonucleotides is schematically presented in (G). The log-normal distribution of vizualized RNA-primed RCA products per cell are presented in (H). The histogram shows the number of individual cells in each subset having specific RCA products within indicated bin ranges.

Article Snippet: The following DNA oligonucleotides were obtained from Metabion: PP5 (5′-TGAGCACAGGGTACTATGCTGCTGCTGTACTACGAGCGGTCTCCAGGAATGCGCATTTTTTTTTTTTGGT); PP6 (5′-AGAGCACAGGGTACTATGCTGCTGCTGTACTACGAGCGGTCTCCAGGAATGCGCATTTTTTTTTTTTGGA); Mva1269I olig. (5′-CCAGGAATGCGCATTTATGT); EM1 (5′-GGTTGAGCACAGGGTACTTTATTGATGG); and DP-FITC (5′-FITC-AATGCTGCTGCTGTACTACGG).

Techniques: In Situ, Ligation, Microscopy, Fluorescence, Labeling