Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Transcriptional Regulation of Endothelial Arginase 2 by Histone Deacetylase 2

doi: 10.1161/atvbaha.114.303685

Figure Lengend Snippet: Figure 2. Histone deacetylase (HDAC) inhibition upregulates arginase 2 (Arg2) activity, mRNA, and protein expression. Human aortic endothelial cells (HAECs) were incubated with 0.4 μmol/L of trichostatin A (TSA) for 18 hours and (A) arginase activity was measured in cell lysates using urea production assay11 and (B) Arg2 protein levels were determined using Western blot (WB). C, Dose-dependent (0, 0.1, 0.3, and 1 μmol/L) and (D) time-dependent (0, 2, 4, 8, and 18 hours) effects of TSA (1 μmol/L) on endothelial Arg2 protein expres- sion were determined using WB. E, HAECs were transduced with 15 MOI of adenoviruses encoding either nontargeted, Arg2 or Arg1 shRNA effect of TSA on total arginase activity was determined using the urea assay. Effect of TSA (1 μmol/L) on Arg2 mRNA levels was determined using (F) RT-polymerase chain reaction (PCR) and (G) real-time PCR in HAECs with increasing doses of TSA (0, 0.1, 0.2, and 1 μmol/L) or nicotinamide 0.1 mmol/L or 0.2 mmol/L (F only, lanes 4 and 5). H, mRNA levels were determined by real-time PCR in iso- lated mice aortas that were incubated with 200 nmol/L TSA for 18 hours. I, Arginase activity was determined from isolated segments of mice aorta exposed to TSA (200 nmol/L). J, Arginase activity was determined from HAECs exposed to TSA (1 μmol/L) or nicotinamide (5 mmol/L) for 30 minutes. *P<0.05 vs control (no TSA), $P<0.05 vs control (no TSA), #P<0.05 vs control (with TSA). WB and RT-PCR experi- ments were performed 3 to 4 times. Fold change in real-time PCR data indicates the differences in the ratio between Arg2 mRNA and 18s mRNA when the TSA-treated and control sample data are measured. IB indicates XXX; and RLU, XXX.

Article Snippet: To define and quantify the nature of TSA modulation of Arg2 expression, human aortic endothelial cells (HAECs) were treated with 400 nmol/L TSA for 18 hours as suggested by the manufacturer (Cell Signaling), and arginase activity and protein expression were then measured.

Techniques: Histone Deacetylase Assay, Inhibition, Activity Assay, Expressing, Incubation, Western Blot, Transduction, shRNA, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Isolation, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Transcriptional Regulation of Endothelial Arginase 2 by Histone Deacetylase 2

doi: 10.1161/atvbaha.114.303685

Figure Lengend Snippet: Figure 4. Effects of trichostatin A (TSA) on arginase transcription and promoter activity. A, Human aortic endothelial cells (HAECs) were pretreated with either 10 μg/mL of the transcriptional inhibitor actinomycin D (Act D) or 10 μg/mL of the protein translation inhibitor cyclo- heximide for 8 hours and cell lysates were then subjected to Western blot (WB) using antibodies against arginase 2 (Arg2) and GAPDH. B, Luciferase activity was determined in HAECs that were transfected with the pGL3 luciferase (LUC) reporter plasmid consisting of the −1 kb human ARG2 promoter and 3 different promoter fragments (as illustrated above the graph) and incubated in the presence of TSA. C, HAECs were transfected with pgL3.4 harboring the 1-kb Arg2 promoter and challenged with increasing doses of TSA. *P<0.05 vs con- trol, and WB results are representatives of 3 independent experiments. IB indicates XXX; and RLU, XXX.

Article Snippet: To define and quantify the nature of TSA modulation of Arg2 expression, human aortic endothelial cells (HAECs) were treated with 400 nmol/L TSA for 18 hours as suggested by the manufacturer (Cell Signaling), and arginase activity and protein expression were then measured.

Techniques: Activity Assay, Western Blot, Luciferase, Transfection, Plasmid Preparation, Incubation

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Transcriptional Regulation of Endothelial Arginase 2 by Histone Deacetylase 2

doi: 10.1161/atvbaha.114.303685

Figure Lengend Snippet: Figure 5. Attenuated nitric oxide production in trichostatin A (TSA)-treated human aortic endothelial cells (HAECs) was restored by arginase inhibition. HAECs were incubated with either TSA (1 μmol/L) alone or together with amino-2-borono-6-hexanoic acid (ABH; 1 mmol/L). A, Representative epifluorescence images and (B) quantitative analysis of DAF-DA fluorescence were used to assess nitric oxide (NO) production. C, A NO analyzer from Siever was used to measure byproducts of NO, mainly nitrite, in HAEC cell media that were incu- bated with either TSA alone or coincubated with TSA and ABH. D, Two hundred ninety-three cells that do not express native endothelial nitric oxide synthase (eNOS) were expressed with constitutively active eNOS constructs and incubated with either TSA alone or together with ABH. The DCFH-DiOxyQ-based fluorescent assay from Cell Biolabs was used to determine NO radicals in cell culture media. *P<0.05 vs control, #P<0.05 vs TSA alone, $P<0.05 vs TSA+ABH. DAF fluorescence images are representative of 3 independent experi- ments. CIeNOS indicates calcium-insensitive endothelial nitric oxide synthase; RFU, XXX; and RNS, XXX.

Article Snippet: To define and quantify the nature of TSA modulation of Arg2 expression, human aortic endothelial cells (HAECs) were treated with 400 nmol/L TSA for 18 hours as suggested by the manufacturer (Cell Signaling), and arginase activity and protein expression were then measured.

Techniques: Inhibition, Incubation, Fluorescence, Construct, Cell Culture, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Transcriptional Regulation of Endothelial Arginase 2 by Histone Deacetylase 2

doi: 10.1161/atvbaha.114.303685

Figure Lengend Snippet: Figure 7. Histone deacetylase (HDAC) 2 is the specific HDAC isoform that regulates arginase 2 (Arg2) expression. Human aortic endothelial cells were transfected with increasing concentrations of (A) HDAC1, (B) HDAC3, (C) HDAC8, or (D) HDAC2 cDNAs, and Arg2 expression was determined by Western blot. E, HAECs that were transfected with HDAC2 siRNA (30 nmol/L) were subjected to immu- noblotting for Arg2 and HDAC2. Results are repre- sentatives of 3 to 4 independent experiments.

Article Snippet: To define and quantify the nature of TSA modulation of Arg2 expression, human aortic endothelial cells (HAECs) were treated with 400 nmol/L TSA for 18 hours as suggested by the manufacturer (Cell Signaling), and arginase activity and protein expression were then measured.

Techniques: Histone Deacetylase Assay, Expressing, Transfection, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Transcriptional Regulation of Endothelial Arginase 2 by Histone Deacetylase 2

doi: 10.1161/atvbaha.114.303685

Figure Lengend Snippet: Figure 8. Increased endothelial arginase 2 (Arg2) expression in response to oxidized low-density lipoprotein (OxLDL) involves promoter activation and histone deacetylase (HDAC) 2. A, Human aortic endothelial cells (HAECs) were exposed to 50 μg/mL of OxLDL for 18 hours and subjected to Western blot (WB) using Arg2 antibody. B, Luciferase activity was determined in HAECs that were transfected with pGL3 luciferase (LUC) reporter plasmids consisting of either −1 kb human ARG2 promoter or 1 of 3 different promoter fragments (as illustrated above the graph in Figure 4B) and incubated with 50 μg/mL of OxLDL for 18 hours. C, HAECs were transfected with an LUC reporter plasmid containing the −1-kb human ARG2 promoter, and cells were exposed to increasing doses of OxLDL (0, 25, 50, and 100 μg/mL) for 18 hours. Firefly luciferase activity was measured and normalized to Renilla luciferase. GFP and HDAC2 adenoviruses were added 8 hours after transfection with the luciferase constructs. D, Binding of HDAC2 to the Arg2 promoter was quantified using a ChIP assay. E, HAECs were exposed to increasing doses of OxLDL (0, 25, 50, and 100 μg/mL) for 18 hours and immunoblotted for HDAC2 and Arg2. F, HAECs with adenoviral-mediated HDAC2 overexpression or controls (Ad-GFP) were exposed to OxLDL, and expression levels of Arg2, HDAC2, and GAPDH (loading control) were determined with WB. *P<0.05 vs control. WB results are representatives of 3 to 4 inde- pendent experiments. IB indicates XXX; and RLU, XXX.

Article Snippet: To define and quantify the nature of TSA modulation of Arg2 expression, human aortic endothelial cells (HAECs) were treated with 400 nmol/L TSA for 18 hours as suggested by the manufacturer (Cell Signaling), and arginase activity and protein expression were then measured.

Techniques: Expressing, Activation Assay, Histone Deacetylase Assay, Western Blot, Luciferase, Activity Assay, Transfection, Incubation, Plasmid Preparation, Construct, Binding Assay, Over Expression, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Transcriptional Regulation of Endothelial Arginase 2 by Histone Deacetylase 2

doi: 10.1161/atvbaha.114.303685

Figure Lengend Snippet: Figure 9. Oxidized low-density lipoprotein (OxLDL)-mediated impairment of vascular relaxation is ameliorated by histone deacetylase (HDAC) 2 overexpression. Isolated mice aortic rings were incubated in EBM2 media containing 100 MOI of either GFP or HDAC2 adeno- viruses. Twenty four hours post transduction, media was replaced with fresh media with or without OxLDL (50 μg/mL). After 48 hours, dose–response effects of acetylcholine (A) and sodium nitroprusside (SNP; B) on vascular relaxation were determined using wire myog- raphy. C, Mice aortic segments were transduced with 100 MOI of either GFP or HDAC2 adenoviruses and subjected to immunoblotting with anti-GFP and anti-HDAC2 antibodies. D, Human aortic endothelial cells (HAECs) overexpressing HDAC2 or GFP were subjected to an arginase activity assay. E, Nitric oxide production was determined using the DAF-FM DA fluorescence assay in HAECs that were transduced with either Ad-Arg2shRNA or Ad-HDAC2shRNA alone, or both reagents in combination. Nontargeted Ad-shRNA was used as a control. *P<0.05 vs control; n=6. RFU indicates XXX; and WT, wild type.

Article Snippet: To define and quantify the nature of TSA modulation of Arg2 expression, human aortic endothelial cells (HAECs) were treated with 400 nmol/L TSA for 18 hours as suggested by the manufacturer (Cell Signaling), and arginase activity and protein expression were then measured.

Techniques: Histone Deacetylase Assay, Over Expression, Isolation, Incubation, Transduction, Western Blot, Arginase Activity Assay, Fluorescence, shRNA, Control

Journal: bioRxiv

Article Title: Characterization of emerging Oropouche virus tropism and pathogenicity

doi: 10.64898/2026.03.25.714204

Figure Lengend Snippet: A: dPCR results of human chondrocytes (HC, left), Fibroblast-Like Synoviocytes (HFLS, middle) and muscular cells (CHQ; right) cultures supernatants infected with OROV at MOI 0.1 and collected at different times (24, 48, 72 h p.i.). The graphs show 18 points (except HFLS with 12 points) per condition, as well as the mean and geometric standard deviation. ns: not significant; *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001 (Dunn-Bonferroni non-parametric test). B: HC (left), HFLS (middle), and CHQ (right) cells were infected with OROV at different MOIs. Cell lysates were collected at 48 or 72h p.i. and analysed by Western blot, using antibodies against calnexin (CNX) as internal control and against the N protein of OROV (OROV N). C: The supernatants from HC (left), HFLS (middle) and CHQ (right) cultures were collected at different times after infection with OROV at MOI 0.1. The viral titer was determined on Vero cells using xCELLigence technology from a standard range. Each condition has 6 replicates. ns: not significant; *: p-value < 0.05; **: p-value < 0.01 (Dunn-Bonferroni non-parametric test).

Article Snippet: Human chondrocytes (HC) and synoviocytes (HFLS) were obtained from Cell Applications, (Ref PB-402-05a and 408K-05a, respectively) and cultured according to manufacturer’s instructions.

Techniques: Infection, Standard Deviation, Western Blot, Control

Journal: bioRxiv

Article Title: Characterization of emerging Oropouche virus tropism and pathogenicity

doi: 10.64898/2026.03.25.714204

Figure Lengend Snippet: HC, HFLS and CHQ cells were infected on coverslips with OROV at MOI 0.1. Gc protein of the OROV virus was detected (green), and nuclei were also labelled (blue). The scale bar represents 200 µm.

Article Snippet: Human chondrocytes (HC) and synoviocytes (HFLS) were obtained from Cell Applications, (Ref PB-402-05a and 408K-05a, respectively) and cultured according to manufacturer’s instructions.

Techniques: Infection, Virus