Journal: Scientific Reports

Article Title: Novel long non-coding RNAs associated with inflammation and macrophage activation in human

doi: 10.1038/s41598-023-30568-1

Figure Lengend Snippet: PMA induced differentiation of THP1 monocytes into macrophages (THP1-Mɸ) and their response to LPS-stimulation. ( A ) THP1 cells (monocytes) were differentiated using PMA (25 nM, 72 h) on a coverslip (35 mm cell culture plate). Cells were immuno-stained with CD68 antibody (mouse) followed by FITC-conjugated secondary antibodies, counterstained with DNA binding dye DAPI, mounted, and analyzed under a fluorescence microscope. Images taken at 40X resolution (bar = 50 μm). ( B ) LPS-stimulation of THP1 (monocytes) and THP1-Mɸ. THP1 and THP1-Mɸ cells were treated with LPS (1 μg/mL, 4 h) independently, total RNA was isolated, reverse transcribed to cDNA, and analyzed by RT-qPCR for expression of IL-6 and IL-1β. Each experiment was repeated at least thrice with three parallel replicates. β-Actin was used as loading control. Data represent mean ± SEM (n = 3); * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: 3 × 10 6 cells THP1 cells were seeded in 60 mm cell culture dishes, differentiated into THP1-MΦ using PMA (as above) and then treated with LPS (1.0 μg/mL, Invivogen) for 4 h (or varying dose or time periods) and subjected to RNA and protein extraction, and immunostaining, as needed , .

Techniques: Cell Culture, Staining, Binding Assay, Fluorescence, Microscopy, Isolation, Reverse Transcription, Quantitative RT-PCR, Expressing, Control

Journal: Scientific Reports

Article Title: Novel long non-coding RNAs associated with inflammation and macrophage activation in human

doi: 10.1038/s41598-023-30568-1

Figure Lengend Snippet: RNAseq analysis of LPS-treated macrophages. THP1 c ells were differentiated into PMA into macrophages (THP1-MΦ), treated with LPS (1.0 μg/mL) for 4 h. Total RNA was extracted from the control cells (C1–C3) and LPS-treated THP1-MΦ cells (L1–L3), quantified and subjected to ribo-depletion followed by library construction using the Illumina TruSeq Stranded Total RNA Library Prep Gold kit. Libraries were sequenced in an Illumina NovaSeq instrument. Differential gene expression analysis was done using the R package edgeR (v3.10.5) (PMID:19910308). Differentially expressed genes (log2 -old) were plotted as a heatmap. Cutoff values of absolute fold change greater than 2.0 and FDR ≤ 0.05 were used to select for differentially expressed genes between sample group comparisons.

Article Snippet: 3 × 10 6 cells THP1 cells were seeded in 60 mm cell culture dishes, differentiated into THP1-MΦ using PMA (as above) and then treated with LPS (1.0 μg/mL, Invivogen) for 4 h (or varying dose or time periods) and subjected to RNA and protein extraction, and immunostaining, as needed , .

Techniques: Control, Gene Expression

Journal: Scientific Reports

Article Title: Novel long non-coding RNAs associated with inflammation and macrophage activation in human

doi: 10.1038/s41598-023-30568-1

Figure Lengend Snippet: Pathways affected by LPS-stimulation of THP1-MΦ. RNAseq data was analysis using Panther-based data analysis to identify different signaling pathways that are affected by LPS-stimulation of macrophages.

Article Snippet: 3 × 10 6 cells THP1 cells were seeded in 60 mm cell culture dishes, differentiated into THP1-MΦ using PMA (as above) and then treated with LPS (1.0 μg/mL, Invivogen) for 4 h (or varying dose or time periods) and subjected to RNA and protein extraction, and immunostaining, as needed , .

Techniques: Protein-Protein interactions

Journal: Scientific Reports

Article Title: Novel long non-coding RNAs associated with inflammation and macrophage activation in human

doi: 10.1038/s41598-023-30568-1

Figure Lengend Snippet: LPS induces inflammation in THP1-macrophages (THP1-Mɸ). THP1-Mɸ cells were treated with LPS (1 μg/mL, 4 h), total RNA and proteins were isolated. RNA was reverse transcribed to cDNA and analyzed by RT-qPCR for expression of proinflammatory cytokines like IL-6 and IL-1β ( A ), as well as top upregulated protein coding genes (found in RNA-seq analysis) including ACOD1 and IDO1 at transcript level ( B ); and hLinfRNAs (1–5) ( C ). ( D ) Western blot analysis of protein coding genes. Proteins from the control and LPS-treated THP1-MΦ were analyzed by Western blot using antibodies against IL6, IL-1β, ACOD1, IDO1, and β-Actin (control). Bands were quantified and plotted in Fig. 4E. The specific region selected for each western blot is shown by red–rectangle in original respective western blot in the supplementary figure . Each experiment was repeated at least thrice with three parallel replicates. β-Actin was used as loading control. Data represent mean ± SEM (n = 3); * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: 3 × 10 6 cells THP1 cells were seeded in 60 mm cell culture dishes, differentiated into THP1-MΦ using PMA (as above) and then treated with LPS (1.0 μg/mL, Invivogen) for 4 h (or varying dose or time periods) and subjected to RNA and protein extraction, and immunostaining, as needed , .

Techniques: Isolation, Reverse Transcription, Quantitative RT-PCR, Expressing, RNA Sequencing, Western Blot, Control

Journal: Scientific Reports

Article Title: Novel long non-coding RNAs associated with inflammation and macrophage activation in human

doi: 10.1038/s41598-023-30568-1

Figure Lengend Snippet: hLinfRNAs are expressed in a dose-dependent manner in THP1-macrophages (THP1-Mɸ) under LPS induced inflammation. THP1-MΦ cells were treated with varying concentration of LPS (0.1- 1000 ng/mL, 4 h), total RNA was isolated and analyzed by RT-qPCR for expression of proinflammatory cytokines (IL6, IL-1β) and top 5 hLinfRNAs. β-Actin was used as loading control. Data represents mean ± SEM (n = 3).

Article Snippet: 3 × 10 6 cells THP1 cells were seeded in 60 mm cell culture dishes, differentiated into THP1-MΦ using PMA (as above) and then treated with LPS (1.0 μg/mL, Invivogen) for 4 h (or varying dose or time periods) and subjected to RNA and protein extraction, and immunostaining, as needed , .

Techniques: Concentration Assay, Isolation, Quantitative RT-PCR, Expressing, Control

Journal: Scientific Reports

Article Title: Novel long non-coding RNAs associated with inflammation and macrophage activation in human

doi: 10.1038/s41598-023-30568-1

Figure Lengend Snippet: Temporal expression of hLinfRNAs under LPS-stimulation of THP1-Mɸ. THP1-Mɸ cells were treated with LPS (1 μg/mL) for varying time periods. RNA was analyzed by RT-qPCR for expression of proinflammatory cytokines (IL6, IL-1β) and top 5 hLinfRNAs. Each experiment was repeated at least thrice with three parallel replicates. β-Actin was used as loading control. Data represents mean ± SEM (n = 3).

Article Snippet: 3 × 10 6 cells THP1 cells were seeded in 60 mm cell culture dishes, differentiated into THP1-MΦ using PMA (as above) and then treated with LPS (1.0 μg/mL, Invivogen) for 4 h (or varying dose or time periods) and subjected to RNA and protein extraction, and immunostaining, as needed , .

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Scientific Reports

Article Title: Novel long non-coding RNAs associated with inflammation and macrophage activation in human

doi: 10.1038/s41598-023-30568-1

Figure Lengend Snippet: hLinfRNAs are regulated by NF-κB signaling pathway in THP1-macrophages. THP1-MΦ cells were treated with IKKβί (SC-514, 25 μM, 1 h) followed by LPS (1 μg/mL). RNA and proteins were isolated from the control and LPS (with and without SC514) -treated cells and analyzed by RT-qPCR and Western blotting respectively. ( A-B ) Western blot analysis for the IκBα, phospho-IκBα, p65 and phospho-p65 (β-actin was used as a loading control). Quantifications are shown in panel 7B. The specific region selected for each western blot are shown by red–rectangle in the original respective western blots, supplementary figure . C-D) RT-qPCR analysis for the expression of pro-inflammatory cytokine (IL6, panel C ) and hLinfRNAs (1–5, panel D ). Data represents mean ± SEM (n = 3). * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: 3 × 10 6 cells THP1 cells were seeded in 60 mm cell culture dishes, differentiated into THP1-MΦ using PMA (as above) and then treated with LPS (1.0 μg/mL, Invivogen) for 4 h (or varying dose or time periods) and subjected to RNA and protein extraction, and immunostaining, as needed , .

Techniques: Isolation, Control, Quantitative RT-PCR, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Novel long non-coding RNAs associated with inflammation and macrophage activation in human

doi: 10.1038/s41598-023-30568-1

Figure Lengend Snippet: Knockdown of hLinfRNA1 down-regulates the LPS-induced inflammatory response in macrophage. THP1-MΦ cells were transfected with hLinfRNA specific antisense oligonucleotide (ASO1 and ASO3) and scramble antisense for 48 h, stimulated with LPS (1 μg/mL) and incubated for additional 4 h. RNA was analyzed by RT-qPCR for expression of hLinfRNA1 and proinflammatory cytokines IL6, TNFα and IL1β (Fig. 8A) and PCR amplified product was analyzed in 2% agarose gel electrophoresis (Fig. 8B). The specific region selected for each agarose gel is shown by red–rectangle in the supplementary figure . Each experiment was repeated at least thrice with three parallel replicates. β-Actin was used as loading control. Data represents mean ± SEM (n = 3); * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: 3 × 10 6 cells THP1 cells were seeded in 60 mm cell culture dishes, differentiated into THP1-MΦ using PMA (as above) and then treated with LPS (1.0 μg/mL, Invivogen) for 4 h (or varying dose or time periods) and subjected to RNA and protein extraction, and immunostaining, as needed , .

Techniques: Knockdown, Transfection, Incubation, Quantitative RT-PCR, Expressing, Amplification, Agarose Gel Electrophoresis, Control

Journal: Clinical and Experimental Dental Research

Article Title: Hydrogen sulfide exposure induces NLRP3 inflammasome‐dependent IL‐1β and IL‐18 secretion in human mononuclear leukocytes in vitro

doi: 10.1002/cre2.69

Figure Lengend Snippet: The IL‐1ß and IL‐18 secretion of three THP1 cell lines exposed to 1 mM NaHS for 24 hr. Prior to NaHS, the cells were exposed to lipopolysaccharides. The THP1‐Null cells showed a statistically higher IL‐1ß and IL‐18 secretion when exposed to NaHS (Mann–Whitney U test, p = .0006 for IL‐1ß and p = .002 for IL‐18) compared with unexposed cells. When the other two cell lines were tested, both unable to form the NLRP3‐inflammasome, there was no difference in IL‐1ß and IL‐18 secretion when exposed to NaHS compared to control. The median of the group is shown as a vertical line

Article Snippet: In contrast, the ASC‐deficient THP1‐defASC cell line and the NLRP3‐deficient human monocytes THP1‐defNLRP3 (also InvivoGen) are both unable to form the NLRP3 inflammasome.

Techniques: MANN-WHITNEY