Journal: Journal of Leukocyte Biology

Article Title: The macrocyclic peptide rhesus theta defensin 1 activates interferon and antiviral pathways in human monocytes

doi: 10.1093/jleuko/qiaf150

Figure Lengend Snippet: Effect of RTD-1 on gene expression in human monocytes. (A) Validation of RNA-seq analysis. qRT-PCR analysis was performed for select genes from monocytes treated with or without RTD-1. (Left) Gene expression was normalized to ACTB expression, and fold change was calculated with respect to control cells (data from 2 control and 2 RTD-1–treated monocyte samples). Blue: control (0.01% HOAC), red: 10 µg/mL RTD-1 treatment. The numbers indicate change in gene expression observed by RNA-seq analysis. (Right) Comparison of fold change observed by RNA-seq and qRT-PCR for genes in the left panel. (B) IPA generated graphical representation of the RTD-1–induced gene expression in human monocytes. The position of the nodes was tweaked for readability. Blue indicates inhibition and orange indicates activation, and the legend for IPA shapes is available at https://qiagen.my.salesforce-sites.com/KnowledgeBase/articles/Knowledge/Legend . (C) GSEA of changes in gene expression induced by RTD-1. ND, not differentially expressed in RNA-seq.

Article Snippet: Cells were treated with 10 μg/mL RTD-1 or with 0.01% HOAc (vehicle control) for 4 h at 37 °C and 5% CO 2 and harvested by centrifugation at 200 g for 8 min. For THP-1, cells were resuspended at 3.3 × 10 5 cells/mL in RPMI + 1% FBS and 1% P/S for 4 h at 37 °C in 5% CO 2 and then treated with 1, 3, or 10 μg/mL RTD-1 or 0.01% HOAc (vehicle) control for 20 h. Cells were then harvested at 200 g for 8 min. RNA was isolated using Quick RNA Miniprep kit (Zymo Research) from 3 experiments.

Techniques: Gene Expression, Biomarker Discovery, RNA Sequencing, Quantitative RT-PCR, Expressing, Control, Comparison, Generated, Inhibition, Activation Assay

Journal: Journal of Leukocyte Biology

Article Title: The macrocyclic peptide rhesus theta defensin 1 activates interferon and antiviral pathways in human monocytes

doi: 10.1093/jleuko/qiaf150

Figure Lengend Snippet: Regulation of gene expression by RTD-1 in THP-1 cells. (A) THP-1 cells were treated with RTD-1 or with 0.01% HOAc vehicle control as indicated and the RNA-seq data from 3 experiments was analyzed by hierarchical clustering. Validation of RNA-seq results using qRT-PCR. Gene expression observed in RNA-seq analysis was evaluated for a few genes using qRT-PCR. The gene expression was normalized to ACTB gene expression and fold change was calculated with respect to the vehicle control. Correlation between qRT-PCR and RNA-seq data (B) with 3 µg/mL RTD-1 and (C)with 10 µg/mL RTD-1.

Article Snippet: Cells were treated with 10 μg/mL RTD-1 or with 0.01% HOAc (vehicle control) for 4 h at 37 °C and 5% CO 2 and harvested by centrifugation at 200 g for 8 min. For THP-1, cells were resuspended at 3.3 × 10 5 cells/mL in RPMI + 1% FBS and 1% P/S for 4 h at 37 °C in 5% CO 2 and then treated with 1, 3, or 10 μg/mL RTD-1 or 0.01% HOAc (vehicle) control for 20 h. Cells were then harvested at 200 g for 8 min. RNA was isolated using Quick RNA Miniprep kit (Zymo Research) from 3 experiments.

Techniques: Gene Expression, Control, RNA Sequencing, Biomarker Discovery, Quantitative RT-PCR

Journal: Journal of Leukocyte Biology

Article Title: The macrocyclic peptide rhesus theta defensin 1 activates interferon and antiviral pathways in human monocytes

doi: 10.1093/jleuko/qiaf150

Figure Lengend Snippet: RTD-1 induced phosphorylation of STAT1. THP-1 cells were pretreated with DMSO or Ruxo for 1 h and then with 0.01% HOAc or RTD-1 as shown. The cells were harvested after 30 min and extracts were analyzed by Western blotting experiments using anti-phospho-STAT1 Y701 antibody or anti-ACTB antibody. (Left) One representative gel from 2 experiments. (Right) Ratio of quantification of the phospho-STAT1 Y701 and ACTB bands from Western blots was plotted with respect to RTD-1 concentration. Results are average of 2 experiments. Error bars indicate standard deviation.

Article Snippet: Cells were treated with 10 μg/mL RTD-1 or with 0.01% HOAc (vehicle control) for 4 h at 37 °C and 5% CO 2 and harvested by centrifugation at 200 g for 8 min. For THP-1, cells were resuspended at 3.3 × 10 5 cells/mL in RPMI + 1% FBS and 1% P/S for 4 h at 37 °C in 5% CO 2 and then treated with 1, 3, or 10 μg/mL RTD-1 or 0.01% HOAc (vehicle) control for 20 h. Cells were then harvested at 200 g for 8 min. RNA was isolated using Quick RNA Miniprep kit (Zymo Research) from 3 experiments.

Techniques: Phospho-proteomics, Western Blot, Concentration Assay, Standard Deviation

Journal: Journal of Leukocyte Biology

Article Title: The macrocyclic peptide rhesus theta defensin 1 activates interferon and antiviral pathways in human monocytes

doi: 10.1093/jleuko/qiaf150

Figure Lengend Snippet: Stimulation of ISRE reporter by RTD-1. The activity of Lucia luciferase secreted in the medium was assayed and the luminescence units are plotted. (A) THP-1 Dual cells were stimulated with HOAc (vehicle) or increasing concentrations of RTD-1. Results are from 3 experiments. * P < 0.05, 2-tailed t test, comparison with HOAc-treated sample. Error bars indicate standard deviation. (B) THP-1 Dual cells were pretreated with DMSO or Ruxo as indicated. Assay done in triplicate. * P < 0.05 when compared with HOAc-treated control, 2-tailed t test. # P < 0.05 when compared with RTD-1 treatment. Error bars indicate standard deviation. (C) THP-1 Dual cells were transferred to RPMI + 1% HI FBS + P/S + normocin medium and either 2.5 μg human IgG1 or anti-IFNAR1 antibodies were added to the culture. Cells were incubated for 2 h, and RTD-1 or IFN-β was added as indicated and the incubation continued for 18 h. Assay was done in triplicate. * P < 0.05 when compared with HOAc-treated control, 2-tailed t test. Error bars indicate standard deviation. (D) THP-1 Dual cells were treated with RTD-1, IFN-β, or both as shown. Assay was done in triplicate. * P < 0.05, 2-tailed t test. Error bars indicate standard deviation.

Article Snippet: Cells were treated with 10 μg/mL RTD-1 or with 0.01% HOAc (vehicle control) for 4 h at 37 °C and 5% CO 2 and harvested by centrifugation at 200 g for 8 min. For THP-1, cells were resuspended at 3.3 × 10 5 cells/mL in RPMI + 1% FBS and 1% P/S for 4 h at 37 °C in 5% CO 2 and then treated with 1, 3, or 10 μg/mL RTD-1 or 0.01% HOAc (vehicle) control for 20 h. Cells were then harvested at 200 g for 8 min. RNA was isolated using Quick RNA Miniprep kit (Zymo Research) from 3 experiments.

Techniques: Activity Assay, Luciferase, Comparison, Standard Deviation, Control, Incubation