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

Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A THP-1 cells were incubated with or without (Ctrl) CLO at different doses for 1 h and then observed with a microscope. B The time-lapse images of THP-1 cells treated with CLO (100 nM). C The hemolytic activity of CLO at different doses was determined. D THP-1 cells were incubated with or without (Ctrl) CLO at different doses for 1 h, and then measured for LDH release. In panels A and B , arrows indicate membrane blebbing, and the scale bar is 30 μm. In panels C and D , values are shown as means ± SD ( N = 3). N, the number of replicates. ** p < 0.01(one-way ANOVA).

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques: Incubation, Microscopy, Activity Assay, Membrane

Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A , B THP-1 cells were treated with CLO (100 nM) or necroptosis inducer TBZ (TNFα, the SMAC mimetic BV-6 and Z-VAD) in the presence of DMSO or the inhibitors GSK’963, GSK’872, and GW806742X (targeting RIPK1, RIPK3, and MLKL respectively) for 1 h or 16 h. The cells were then subjected to LDH release determination ( A ) and microscopy after PI staining ( B ). Scale bar, 30 μm. C THP-1 cells were treated with or without (Ctrl) CLO (100 nM) in the presence of Q-VD-OPh, Ac-YVAD-CMK, Ac-DEVD-CMK, Z-LEVD-FMK, Z-IETD-FMK, or DMSO for 1 h. LDH release was then measured. D THP-1 cells were treated with or without (Ctrl) CLO (100 nM) in the presence or absence of different concentrations of Ac-YVAD-CMK for 1 h. LDH release was then measured. E , F THP-1 WT and THP-1 GSDMD-KO cells were treated with or without (Ctrl) CLO (100 nM) or nigericin (Nig) for 1 h. LDH ( E ) and IL-1β ( F ) release was then determined. G THP-1 GSDMD-KO cells were treated as C and then measured for LDH release. ** p < 0.01. NS no significance (one-way ANOVA test A , C , D , and G or student’s unpaired t test E and F . Values are shown as means ± SD ( N = 3). N the number of replicates.

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques: Microscopy, Staining

Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A J774A.1 cells were pretreated with MCC950, VX765, or DMSO for 1 h and then treated with CLO (CLO 100 nM) or ATP for 1 h. LDH release was then determined. B , C The cell lysate and supernatants from J774A.1 cells treated as above was immunoblotted with antibodies against Casp1, GSDMD, or β-actin (loading control). D , E PMA-differentiated THP-1 cells with or without (Null) deficiency in NLRP3 (NLRP3-KD) or Casp1 (Casp1-KD) were treated with or without (Ctrl) CLO (CLO 100 nM) or nigericin (Nig) for 1 h. LDH ( D ) and IL-1β ( E ) release was then determined. F The supernatant and the corresponding cell lysate from the above ( D , E ) treated J774A.1 cells were blotted with antibodies against Casp1, GSDMD, or β-actin (loading control). For panels A , D , and E , values are shown as means ± SD ( N = 3). N, the number of replicates. ** p < 0.01 (one-way ANOVA).

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques:

Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A , B J774A.1 cells pretreated with DCFH-DA were incubated with or without (Ctrl) DPI or NAC for 1 h. The cells were treated with or without CLO (10 nM) for 30 min. ROS production ( A ) and fluorescence intensity (λex, 488 nm; λem, 525 nm) ( B ) were then determined. C – G J774A.1 cells were pretreated with or without (−) DPI or NAC for 1 h and then treated with CLO (10 nM) or ATP for 1 h. Cell viability ( C ), LDH release ( D ), and IL-1β ( E ) release were determined. ASC speck (red) was detected by treating the cells with ASC-antibody and DAPI ( F ). The supernatant plus the corresponding cell lysate were blotted with antibody against Casp1, GSDMD, or β-actin (loading control) ( G ). H J774A.1 cells were incubated with or without (Ctrl) CLO (100 nM) or nigericin (Nig) for 30 min, and intracellular K + was then determined. I – K J774A.1 cells were pretreated with or without (−) different concentrations of KCl for 1 h, and then treated with or without (−) CLO (100 nM) for 1 h. LDH ( I ) and IL-1β release ( J ) was then determined. Immunoblot analysis of Casp-1, GSDMD, or β-actin was performed as above ( K ). Scale bars of panels A and F are 30 μm and 10 μm, respectively. For panels B – E and H – J , values are shown as means ± SD ( N = 3). N, the number of replicates. ** p < 0.01, * p < 0.05. NS no significance (one-way ANOVA).

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques: Incubation, Fluorescence, Western Blot

Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A CLO was incubated with a membrane lipid strip spotted with 15 lipids, and the bound CLO was detected by immunoblotting. B THP-1 cells were incubated with or without (Ctrl) CLO (100 nM) or cholesterol (Cho.)-pretreated CLO for 1 h. CLO was localized by immunofluorescence microscopy using dyLight 650 anti-6×His tag antibody. Scale bar, 30 μm. C – E J774A.1 cells were treated with or without (Ctrl) CLO (100 nM), nigericin (Nig), or Cho-pretreated CLO or Nig for 1 h. LDH ( C ) and IL-1β ( D ) release was then determined, and Casp1 and GSDMD cleavage was determined by Western blot with antibodies against Casp1, GSDMD, and β-actin (loading control) ( E ). For panels C and D , values are shown as means ± SD ( N = 3). N, the number of replicates. ** p < 0.01. NS no significance (student’s unpaired t test).

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques: Incubation, Membrane, Stripping Membranes, Western Blot, Immunofluorescence, Microscopy

Journal: Cell Death Discovery

Article Title: Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

doi: 10.1038/s41420-024-01887-7

Figure Lengend Snippet: A J774A.1 cells were treated with CLO (100 nM) or its mutants (100 nM) for 1 h, and LDH release was then determined. B Sterile defidrinated sheep blood was incubated with CLO (100 nM) or its mutants (100 nM) for 30 min and then detected for hemolysis. C A membrane lipid strip was incubated with the W477S-W479S mutant, and the bound protein was detected by immunoblotting. D THP-1 cells were incubated with or without (Ctrl) CLO (100 nM) or the W477S-W479S mutant (100 nM) for 1 h. The cells were stained with DAPI and subjected to immunofluorescence microscopy with dyLight 650 anti-6×His tag antibody. Scale bar, 30 μm. E , F J774A.1 cells were treated with or without (Ctrl) ATP, CLO (100 nM), or the W477S-W479S mutant (100 nM) for 1 h. The cells were determined for IL-1β release ( E ) and Casp1/GSDMD cleavage by immunoblot using antibodies against Casp1, GSDMD, and β-actin (loading control) ( F ). J774A.1 cells were treated with mutants (100 nM) for 1 h. LDH ( G ), IL-1β ( H ) and immunoblot analysis of Casp-1 and GSDMD ( I ) were assessed as above. ** p < 0.01. NS no significance (one-way ANOVA test A , B , G , and H or student’s unpaired t test E . Values are shown as means ± SD ( N = 3). N the number of replicates.

Article Snippet: THP-1-Null, THP-1-defCasp1 (Casp1-KD), and THP-1-defNLRP3 (NLRP3-KD) were purchased from InvivoGen and cultured as instructed by the manufacturer.

Techniques: Sterility, Incubation, Membrane, Stripping Membranes, Mutagenesis, Western Blot, Staining, Immunofluorescence, Microscopy

Journal: iScience

Article Title: Chemical and biological characterization of vaccine adjuvant QS-21 produced via plant cell culture

doi: 10.1016/j.isci.2024.109006

Figure Lengend Snippet: In vitro biological characterization; ccQS-21 and beQS-21 equivalently induce hemolysis, trigger the innate immune cell stress response, and enhance antigen processing and presentation to enhance the potency of the vaccinal immune synapse (A) Upon incubation with human red blood cells both beQS-21 and ccQS-21 retain equivalent saponin-specific hemolytic properties at pH-7.4 with matching half maximal effective concentration (EC50). (B) Upon pre-conditioning of THP1 human myeloid cells with a TLR4 agonist (LPS), both beQS-21 and ccQS-21 induce inflammasome-dependent secretion of IL-1β and the production of alarmins associated with pyroptosis such as HMGB1. Both IL-1β secretion and pyroptosis can be regulated by the canonical inflammasome pathway and can be suppressed by the disruption of the genes encoding for NLRP3, ASC and Caspase-1 or by the use of Caspase-1 inhibitor, ZVAD. (C) Both beQS-21 and ccQS-21 can comparably induce a broader inflammatory response in vitro . The breadth of the inflammatory response induced by the stimulation of human granulocyte macrophage colony stimulating factor (GMCSF) and IL-4 differentiated Monocyte derived Dendritic Cells (Mo-DCs) using both QS-21 was assessed with multiplex cytokine quantification. Both beQS-21 (BE) and ccQS-21 (CC) induce the same inflammatory signature which is further enhanced by formulating with TLR4 agonist. (D) Pseudo cross-presentation assay using cell viability to demonstrate that beQS-21 and ccQS-21 equivalently induce cytotoxicity through the regulation of the cytosolic translocation from the phagolysosome (e.g., as of the indicated toxin: saporin), a critical process for antigen cross presentation. (E) Flow cytometry analysis demonstrating that both QS-21 equivalently enhance large antigen processing and cross-presentation on MHC-I using a T cell receptor (TCR)-like antibody against the H-2K b -SIINFEKL complex. (F) In vitro antigen cross-presentation assay demonstrating that ccQS-21 and beQS-21 equivalently enhance antigen processing and minimal epitope cross-presentation to activate transgenic T cells with matched OT1 TCR to express NFAT luciferase reporter. (G) OT1 T cell proliferation shows ccQS-21 and beQS-21 equivalently enhance antigen cross-presentation to induce the proliferation of primary T cells with matched OT1 TCR. OVA: Ovalbumin, OT1 long peptide: 29mer peptide including SIINFEKL, BFM: Bafilomycin A1, inhibiting the vacuolar type H + -ATPase (v-ATPase) to transfer protons into the lysosome.

Article Snippet: THP1-Null2, THP1-KO-ASC, THP1-KO-NLRP3, THP1-def CASP1, THP1-Null, THP1-HMGB1-Lucia, HEK-Blue- IL-1b cell lines were purchased from Invivogen and maintained following the vendor recommendation.

Techniques: In Vitro, Incubation, Concentration Assay, Disruption, Derivative Assay, Multiplex Assay, Translocation Assay, Flow Cytometry, Transgenic Assay, Luciferase

Journal: iScience

Article Title: Chemical and biological characterization of vaccine adjuvant QS-21 produced via plant cell culture

doi: 10.1016/j.isci.2024.109006

Figure Lengend Snippet:

Article Snippet: THP1-Null2, THP1-KO-ASC, THP1-KO-NLRP3, THP1-def CASP1, THP1-Null, THP1-HMGB1-Lucia, HEK-Blue- IL-1b cell lines were purchased from Invivogen and maintained following the vendor recommendation.

Techniques: Recombinant, Cell Isolation, Enzyme-linked Immunospot, Cytotoxicity Assay, Luciferase, Expressing