Journal: Hypertension research : official journal of the Japanese Society of Hypertension

Article Title: Histone deacetyltransferase inhibitors Trichostatin A and Mocetinostat differentially regulate MMP9, IL-18 and RECK expression, and attenuate Angiotensin II-induced cardiac fibroblast migration and proliferation.

doi: 10.1038/hr.2016.54

Figure Lengend Snippet: Figure 1 Silencing HDAC1 and HDAC2 inhibits Ang-II-induced cardiac fibroblast proliferation and migration. (a) Expression of HDAC1 and HDAC2 at basal conditions was analyzed by immunoblotting. CF1, CF2: two independent cultures of primary cardiac fibroblasts. Cell extracts from human epithelial cells (HeLa) and mouse embryonic fibroblasts (NIH3T3) served as positive controls (n = 3). (b) Effect of Ang-II dose on activation of HDAC1 and HDAC2 in quiescent CF as analyzed by a colorimetric assay using nuclear protein extracts (100 μg) immunoprecipitated with HDAC1- or HDAC2-specific antibodies. Deacetylase activity was calculated in pmoles min −1 mg−1. Results are expressed as fold change from controls (n = 6). (c) Silencing HDAC1 or HDAC2 attenuates Ang-II-induced CF proliferation. CF transduced with lentiviral HDAC1 or HDAC2 shRNA (moi 0.5 for 24) were treated with Ang-II (10 −7 M) for 48 h, and proliferation analyzed by CyQUANT assay. (d) Silencing HDAC1 or HDAC2 inhibits Ang-II (10 −7 M for 12 h) -induced CF migration. Migration was analyzed using BioCoat Matrigel invasion chambers and MTT assay. (e) Knockdown of HDAC1 and HDAC2 was confirmed by immunoblotting (n = 3). Akt served as an off-target. (f) Silencing HDAC1 or HDAC2 failed to induce cell death. Forty-eight hours after lentiviral transduction, cell death was analyzed by an ELISA that quantifies mono and oligonucleosomal fragmented DNA in cytoplasmic extracts. (b, c, d, f) *Poat least 0.01 vs. untreated, †Poat least 0.05 vs. Ang-II (n = 6-12).

Article Snippet: Anti-vimentin (#sc-373717) antibodies used in fibroblast characterization, anti-caspase-3 (35 kDa, #sc-136219) antibodies and lentiviral GFP shRNA (#sc-45924-V) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Migration, Expressing, Western Blot, Activation Assay, Colorimetric Assay, Immunoprecipitation, Histone Deacetylase Assay, Activity Assay, Transduction, shRNA, CyQUANT Assay, MTT Assay, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Oncology

Article Title: HLA-BAT1 alters migration, invasion and pro-inflammatory cytokines in prostate cancer

doi: 10.3389/fonc.2022.969396

Figure Lengend Snippet: BAT1 Expression was decreased after siRNA Transfection and increased after cDNA Transfection in PC3 and 22RV1 cells. The samples were collected for a period of 24 hours. (A) Representative images and quantification of BAT1 protein expression in PC3 prostate cancer cells using western blot analysis showed a significant decrease in siBAT1 transfected cells when compared to control (*P<0.05). (B) Quantification of BAT1 RNA expression using RT-PCR in PC3 prostate cancer cells showed a significant decrease in BAT1 expression in siBAT1 transfected cells when compared to control (****P < 0.0001). (C) Representative images and quantification of BAT1 protein expression in PC3 prostate cancer cells using western blot analysis showed a significant increase in protein expression in BAT1cDNA when compared to control (***P < 0.001). (D) Quantification of BAT1 RNA expression using RT-PCR in PC3 prostate cancer cells showed a significant increase in BAT1 protein expression in BAT1cDNA cells when compared to control (**P < 0.01). (E) Representative images and quantification of BAT1 protein expression in 22RV1 prostate cancer cells using western blot analysis showed a significant decrease in siBAT1 transfected cells when compared to control (**P < 0.01). (F) Quantification of BAT1 RNA expression using RT-PCR in 22RV1 prostate cancer cells showed a significant decrease in BAT1 expression in siBAT1 transfected cells when compared to control (**P < 0.01) (G) Representative images and quantification of BAT1 protein expression in 22RV1 prostate cancer cells using western blot analysis showed a significant increase in BAT1cDNA when compared to control (***P < 0.001). (H) Quantification of BAT1 RNA expression using RT-PCR in 22RV1 prostate cancer cells showed a significant increase in BAT1 protein expression in BAT1cDNA cells when compared to control (**P < 0.01).

Article Snippet: Prostate cancer cells, PC3 and 22RV1, were seeded and transfected with BAT cDNA-GFP tagged lentiviral particles (BAT1cDNA) (pLenti-C-mGFP vector) (Origene, Rockville, MD, USA) using Polybrene transfection reagent (EMD Millipore, Burlington, MA) and OPTI-MEM Reduced Serum Medium.

Techniques: Expressing, Transfection, Western Blot, RNA Expression, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Oncology

Article Title: HLA-BAT1 alters migration, invasion and pro-inflammatory cytokines in prostate cancer

doi: 10.3389/fonc.2022.969396

Figure Lengend Snippet: BAT1 down-regulation increased PC3 prostate cancer cell migration and BAT1 overexpression decreased PC3 prostate cancer cell migration. (A) Representative images of Control PC3 and siBAT1 PC3 cells using 4X magnification. (B) Relative invasion of siBAT1 PC3 cells caused a significant increase in migration at 12hrs (**P < 0.01) and 24hrs (*P < 0.05) when compared to control. (C) Representative images of Control PC3 and BAT1cDNA PC3 cells using 4X magnification. (D) Relative invasion of (+) BAT1cDNA PC3 cells caused a significant decrease in migration at 12hrs (**P < 0.01) and 24hrs (***P < 0.001) when compared to control.

Article Snippet: Prostate cancer cells, PC3 and 22RV1, were seeded and transfected with BAT cDNA-GFP tagged lentiviral particles (BAT1cDNA) (pLenti-C-mGFP vector) (Origene, Rockville, MD, USA) using Polybrene transfection reagent (EMD Millipore, Burlington, MA) and OPTI-MEM Reduced Serum Medium.

Techniques: Migration, Over Expression

Journal: Frontiers in Oncology

Article Title: HLA-BAT1 alters migration, invasion and pro-inflammatory cytokines in prostate cancer

doi: 10.3389/fonc.2022.969396

Figure Lengend Snippet: BAT1 down-regulation increased PC3 and 22RV1 prostate cancer cell and BAT1 overexpression decreased PC3 and 22RV1 prostate cancer cell invasion. (A, B) Representative images of invasive siBAT1 PC3 and 22RV1 cells at a 4X magnification. (C) Relative invasion of siBAT1 PC3 cells caused a significant increase in invasion when compared to control at 12hrs (**P < 0.01) and 24hrs (****P < 0.0001). (D) Relative invasion of siBAT1 22RV1 cells caused a significant increase in invasion when compared to control at 12hrs (*P < 0.05) and 24hrs (***P < 0.001). (E, F) Representative images of invasive BAT1cDNA PC3 and 22RV1 cells at a 4X magnification. (G) Relative invasion of BAT1cDNA PC3 cells caused a significant decrease in invasion when compared to control at 12hrs (****P < 0.0001) and 24hrs (****P < 0.0001). (H) Relative invasion of BAT1cDNA 22RV1 cells caused a significant decrease in invasion when compared to control at 12hrs (***P < 0.001) and 24hrs (***P < 0.001).

Article Snippet: Prostate cancer cells, PC3 and 22RV1, were seeded and transfected with BAT cDNA-GFP tagged lentiviral particles (BAT1cDNA) (pLenti-C-mGFP vector) (Origene, Rockville, MD, USA) using Polybrene transfection reagent (EMD Millipore, Burlington, MA) and OPTI-MEM Reduced Serum Medium.

Techniques: Over Expression

Journal: Frontiers in Oncology

Article Title: HLA-BAT1 alters migration, invasion and pro-inflammatory cytokines in prostate cancer

doi: 10.3389/fonc.2022.969396

Figure Lengend Snippet: BAT1 expression showed changes in genes associated with inflammation, adhesion, and metastasis in PC3 and 22RV1 cells using qRT-PCR. The samples were collected for a period of 24 hours. (A, C) BAT1cDNA PC3 and 22RV1 cells showed a significant decrease in TNF-α expression when compared to siBAT1 (****P < 0.0001). (B, D) BAT1cDNA PC3 and 22RV1 cells showed a significant decrease in IL-6 expression when compared to siBAT1 (**P < 0.01) (***P < 0.001). (E) BAT1cDNA PC3 cells showed a significant decrease in MMP13 expression when compared to siBAT1 (****P < 0.0001). (F) BAT1cDNA 22RV1 cells showed a significant decrease in MMP10 expression when compared to siBAT1 (**P < 0.01). (G) BAT1cDNA 22RV1 cells showed a significant increase in TIMP2 expression when compared to siBAT1 (***P < 0.001).

Article Snippet: Prostate cancer cells, PC3 and 22RV1, were seeded and transfected with BAT cDNA-GFP tagged lentiviral particles (BAT1cDNA) (pLenti-C-mGFP vector) (Origene, Rockville, MD, USA) using Polybrene transfection reagent (EMD Millipore, Burlington, MA) and OPTI-MEM Reduced Serum Medium.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: HLA-BAT1 alters migration, invasion and pro-inflammatory cytokines in prostate cancer

doi: 10.3389/fonc.2022.969396

Figure Lengend Snippet: In vivo expression of BAT1, Ki67, TNF-α, IL-6, and MMP10. All of the samples were collected for a period of 24 hours. (A) Representative images of immunohistochemical staining BAT1 expression in mice prostate tumors previously injected with 22RV1 cells, BAT1cDNA 22RV1 cells, or shBAT1 22RV1 cells at a 20X (50μm) or 60X magnification (10μm) magnification. (B) Quantification mice prostate tumors obtained from BAT1cDNA mice prostate tumors showed a significant increase in BAT1 expression using immunohistochemistry when compared to control (*P < 0.05) and shBAT1 (**P < 0.01). (C) shBAT1 (**P < 0.01) and BAT1cDNA (*P < 0.05) tumors showed a significant decrease in Ki67 expression when compared to control. (D) shBAT1 tumors showed a significant increase in TNF-α expression when compared to control and BAT1cDNA (*P < 0.05). (E) shBAT1 tumors showed a significant increase in IL-6 expression when compared to control and BAT1cDNA mice prostate tumors (*P < 0.05). (F) shBAT1 tumors showed a significant increase in MMP10 expression when compared to control (*P < 0.05).

Article Snippet: Prostate cancer cells, PC3 and 22RV1, were seeded and transfected with BAT cDNA-GFP tagged lentiviral particles (BAT1cDNA) (pLenti-C-mGFP vector) (Origene, Rockville, MD, USA) using Polybrene transfection reagent (EMD Millipore, Burlington, MA) and OPTI-MEM Reduced Serum Medium.

Techniques: In Vivo, Expressing, Immunohistochemical staining, Staining, Injection, Immunohistochemistry

Journal: F1000Research

Article Title: A co-culture model of the bovine alveolus

doi: 10.12688/f1000research.18696.2

Figure Lengend Snippet: List of reagents required for isolation of alveolar type II cells, generation of lentivirus and assembly of co-culture model.

Article Snippet: SV40 Lentivirus particles , AMS Biotechnology , LVP557-GP.

Techniques: Isolation, Plasmid Preparation, Expressing, Clone Assay, Transfection, Modification

Journal: F1000Research

Article Title: A co-culture model of the bovine alveolus

doi: 10.12688/f1000research.18696.2

Figure Lengend Snippet: For karyotype analysis, a metaphase spread was performed on Colcemid cell cycle arrested cultures for both BATII and B2AE, comparing with the wild-type ATII ( A ). The Bmi1 / hTERT transduced B2AE cell line was found to maintain the wild type bovine karyotype of 2n = 60, whilst the SV40 / hTERT transduced BATII cell line showed signs of genomic instability at both passages 14 and 23. This was reflected in a growth curve analysis of the two cell lines, which was performed by seeding primary ATII cells into a 96 well plate, comparing over 9 days with the BATII and B2AE cell lines ( B ), using the Cell Titre Aqueous One Assay (Promega). Later passages (p10 and p23) of BATII cells exhibited a considerably higher rate of proliferation than the wild type ATII, whilst the B2AE cell line at all passages was comparable with its wild type counterpart. Data shown as optical density read at 490 nm (OD 490nm ); Mean ± SD, n=3.

Article Snippet: SV40 Lentivirus particles , AMS Biotechnology , LVP557-GP.

Techniques:

Journal: F1000Research

Article Title: A co-culture model of the bovine alveolus

doi: 10.12688/f1000research.18696.2

Figure Lengend Snippet: Light microscopy images of the wild type ATII, SV40 / hTERT lentivirus transduced BATII and Bmi1 / hTERT lentivirus transduced B2AE were generated from cultures seeded into T75 cm 2 flasks and imaged on a Nikon Eclipse Ci upright microscope ( A ). Scale bar 50 μm. For IF, each cell type was seeded onto a Nunc Lab Tek II coverglass, before fixation and staining with the ATII marker SLC434A2 and vimentin, a marker of epithelial-mesenchymal transition ( B ). Both cell lines were more prone to colony formation than their wild-type counterpart, with BATII cells in particular forming 3D structures, whilst B2AE frequently formed ring-like colonies around a central ‘lumen’. Cells which exhibited a flattened morphology on the exterior of the colonies of both cell lines stained most strongly for vimentin, with one lone cell in the BATII image notable for the complete absence of SLC34A2 (middle panel, white arrow), which remained strong in the centre of the 3D colony. Scale bar = 100 μm.

Article Snippet: SV40 Lentivirus particles , AMS Biotechnology , LVP557-GP.

Techniques: Light Microscopy, Generated, Microscopy, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Polycystin-1 Enhances Stemmness Potential of Umbilical Cord Blood-Derived Mesenchymal Stem Cells

doi: 10.3390/ijms22094868

Figure Lengend Snippet: Validation of PKD1 expression in transcript and protein level by PKD1 -expressing GFP/lentiviral vector. ( A ) GFP expression in UCB-MSC transfected with PKD1 -expressing vector. PKD1 expression ( B ) and quantitative data ( C ) in transcript level of PKD1 expressing UCB-MSC; ( D ) The expression of PKD1 at the protein level. Data shown represent the mean ± SD ( n = 3). ** p < 0.02 or *** p < 0.01 versus UCB-MSC only group.

Article Snippet: Each cell line was transfected with PKD1 overexpressing plasmid in the lentiviral GFP vector (Origene, Rockville, MD, USA) using TransIT-X2 (Mirus Bio, Madison, WI, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Plasmid Preparation, Transfection