Journal: bioRxiv

Article Title: Chloride Homeostasis Regulates cGAS-STING Signaling

doi: 10.1101/2024.04.08.588475

Figure Lengend Snippet: ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal fibroblasts (HDF) were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.

Article Snippet: THP1-Blue™ ISG cells were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Activation Assay, Cell Culture, Activity Assay, Transfection, Comparison, Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: bioRxiv

Article Title: Chloride Homeostasis Regulates cGAS-STING Signaling

doi: 10.1101/2024.04.08.588475

Figure Lengend Snippet: (a) RAW-Dual cells were pretreated with 100 µM 2,3-cGAMP for 30 min, followed by washing with PBS, and then incubated with 100 μM NPPB and 15 μM H-151 overnight. (b) RAW-Dual cells were pretreated with 100 µM DMXAA or 100 µM 2,3-cGAMP for 30 min, followed by washing with PBS, and then incubated with 150 μM DIDS, and 15 μM H-151 overnight. (c) THP1-Blue ISG cells were pretreated with 100 μM NPPB, 200 µM IAA-94, 100 μM FFA, and 15 μM H-151 for 1 h and then stimulated with 30 µM MSA-2 overnight. (d) IFN-β mRNA expression levels were measured in THP1 cells pretreated with 100 μM NPPB, and 150 μM DIDS overnight and stimulated with 100 µM 2,3-cGAMP overnight. mRNA levels were measured using the RT-qPCR. (e) IFN-β mRNA expression levels were measured in THP1 cells pretreated with 100 μM NPPB, 150 μM DIDS and 15 μM H-151 overnight and stimulated with 30 µM MSA-2 overnight. mRNA levels were measured using the RT-qPCR. (a-e) Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. (f-g) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with ( f ) 100 μM NPPB, ( g ) 150 μM DIDS, and 15 μM H-151 overnight and then stimulated with 100 µM 2,3-cGAMP overnight. Experiments were performed in three biological replicates. (h) Immunofluorescent analysis of HDF cells pretreated with 100 μM NPPB, and 150 μM DIDS overnight and then stimulated with 30 µM MSA-2 for 6 h. (i) Percentage of cells with STING puncta correlating to ( h ). Experiments were performed in three biological replicates (>125 cells per trial). Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison.

Article Snippet: THP1-Blue™ ISG cells were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Incubation, Expressing, Quantitative RT-PCR, Comparison, Western Blot

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: Communications Biology

Article Title: Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins

doi: 10.1038/s42003-021-02953-x

Figure Lengend Snippet: a HT3 B3-WT (blue) or HT3 B3-KO (red) cells were subjected to nucleofection (nuc, +) in the absence (−) or presence (+) of 5 ug/ml LPS-EK (LPS), caspase-1 inhibitor, VX765, or lysosomal cysteine protease inhibitor, E64. An aliquot of cells were also not nucleofected (nuc, −). After 3 h, cells were stained with SG and Hoescht 33342 and imaged. Quantification: (# Sytox positive nuclei/# of blue nuclei) × 100. Means ± SD were compared using a two-tailed t -test (* P < 0.05). b Immunoblot of gasdermin D (GSDMD) and gasdermin E (GSDME) cleavage in THP1 cells treated with α-hemolysin (HlA); positive control. HT3 B3-WT /HT3 B3-KO were nucleofected with 0, 1, or 5 µg of LPS and left to recover for 1 h. Open arrowheads: full-length GSDMD and GSDME based on molecular mass. Black arrowheads: cleaved GSDMD. Dashed box: area contrast enhanced for clarity. Note, the positive control for GSDME cleavage is provided in Fig. . c , d Flow cytometry analysis of HT3 B3-WT /HT3 B3-KO cells treated with a lysosomotropic agent, LLOMe ( c ) or 1 µg/ml LPS ( d ). Cells were stained with the lysosomotropic dye, acridine orange (Y-axis), and the cell viability dye, Sytox™ blue (X-axis). Lines indicate the threshold fluorescence levels (gate) to determine each quadrant. Numbers indicate cell percentage in each quadrant. e Schematic representation of each quadrant. Quadrant 1 (Q1) contained live cells positive for lysosomal staining (acridine orange positive, Sytox blue negative). Quadrant 2 (Q2) contained dead cells with positive lysosomal staining (acridine orange positive, Sytox blue positive). Quadrant 3 (Q3) contained dead cells negative for lysosomal staining (acridine orange negative, Sytox blue positive). Quadrant 4 (Q4) contained live cells negative for lysosomal staining (acridine orange negative, Sytox blue negative). Arrows indicate the timing of fluorescence loss for different cell death pathways. If the lysosomal loss occurred prior to plasma membrane permeabilization, then the percentage of cells will increase from Q1 to Q4 to Q3 over time. If the lysosomal loss occurred after plasma membrane loss, then the percentage of cells will increase from Q1 to Q2 to Q3 over time. f , g Graphical representation of the average of three separate experiments indicating the percentage of cells treated with LLOMe ( f ) or nucleofected LPS ( g ) in HT3 B3-WT (blue) and HT3 B3-KO (red) cells. Error bars represent means ± SD. Uncropped immunoblots can be found in Supplementary Fig. .

Article Snippet: The human monocyte cell line THP1-HMGB1-Lucia™ (thp-gb1lc) was obtained from InvivoGen.

Techniques: Protease Inhibitor, Staining, Two Tailed Test, Western Blot, Positive Control, Flow Cytometry, Fluorescence, Membrane