Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: STING1 is required for LPS-induced ACOD1 expression (A–D) RAW264.7 and THP1 cells were treated with LPS (50-5000 ng/mL) in the absence or presence of 3′3′-cGAMP (10 μg/mL) for 6 h, and then Acod1/ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (E and F) RAW264.7 and THP1 cells were treated with LPS (500 ng/mL) in the absence or presence of 2′2′-cGAMP, 2′3′-cGAMP, c-di-AMP, or c-di-GMP at 10 μg/mL for 6 h, and then Acod1/ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; p < 0.01 versus LPS along group; two-way ANOVA with Tukey’s multiple comparisons test). (G–J) WT and V155M-THP1 cells were treated with indicated LPS for 6 h, and then IFNA1 mRNA, IL6 mRNA, ACOD1 mRNA, and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (K) Western blot analysis of protein expression in indicated THP1 cells following treatment with LPS (500 ng/mL) for 6 h. (L) In parallel, intracellular itaconate concentration was assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: STING1-V155M THP1 , InvivoGen , thpd-m155.

Techniques: Expressing, Concentration Assay, Western Blot

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: MYD88, but not CGAS, mediates LPS-induced ACOD1 expression (A–C) Indicated THP1 cells were treated with LPS (500 ng/mL) in the absence or presence of 3′3′-cGAMP (10 μg/mL), G3-YSD (1 μg/mL), G3-YSD-Ctrl (1 μg/mL), or LyoVec for 6 h, and then ACOD1 or IFNA1 mRNA was assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (D and E) Indicated THP1 cells were treated with LPS (500 ng/mL) in the absence or presence of 3′3′-cGAMP, 2′2′-cGAMP, 2′3′-cGAMP, c-di-AMP, or c-di-GMP at 10 μg/mL for 6 h, and then ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (F) Western blot analysis of protein expression in indicated V155M-THP1 cells following treatment with LPS (500 ng/mL) for 6 h. (G and H) Analysis of ACOD1 mRNA and intracellular itaconate concentration in indicated V155M-THP1 cells following treatment with LPS (50-5000 ng/mL) for 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: STING1-V155M THP1 , InvivoGen , thpd-m155.

Techniques: Expressing, Concentration Assay, Western Blot

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: The MYD88-STING1 protein complex prevents autophagic degradation of STING1 (A) IP analysis of the MYD88-STING1 protein complex in THP1 cells following treatment with LPS (500 ng/mL) and 3′3′-cGAMP (10 μg/mL) for 6 h. (B) Representative colocalization images of MYD88 and STING1 in RAW264.7 cells in the presence or absence of LPS (500 ng/mL) and 3′3'-cGAMP (10 μg/mL) for 6 h. Bar = 10 μm. (C–F) Western blot analysis of protein expression in indicated THP1 cells following treatment with LPS (500 ng/mL) and 3′3′-cGAMP (10 μg/mL) in the absence or presence of chloroquine (50 μM) or MG132 (5 μM) for 6 h. (G) qPCR analysis of ACOD1 mRNA expression in indicated THP1 cells following treatment with LPS (500 ng/mL) and 3′3′-cGAMP (10 μg/mL) in the absence or presence of chloroquine (CQ; 50 μM) for 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: STING1-V155M THP1 , InvivoGen , thpd-m155.

Techniques: Western Blot, Expressing

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: STING1 and MYD88 selectively mediate TLR signaling to induce ACOD1 expression (A) Indicated THP1 cells were stimulated with pam3CSK4 (1 ng/mL), HKLM (10 7 cells/mL), poly (I:C) (10 μg/mL), LPS (500 ng/mL), FLA-ST (100 ng/mL), FSL1 (0.1 ng/mL), imiquimod (5 μg/mL), ssRNA40 (5 μg/mL), or ODN2006 (10 μg/mL) for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (B) Wild-type THP1 cells were stimulated with indicated TLR ligands for 3 h and the mRNA level of TNF was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples). (C) Wild-type THP1 cells were stimulated with indicated TLR ligands in the absence or presence of 3′3′-cGAMP (10 μg/mL) for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (D) Wild-type and V115M THP1 cells were stimulated with indicated TLR ligands for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). The TLR ligand concentration used in panels B-D is the same as for panel A.

Article Snippet: STING1-V155M THP1 , InvivoGen , thpd-m155.

Techniques: Expressing, Concentration Assay

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: The IRF3-JUN transcription factor complex is required for ACOD1 upregulation (A) Western blot analysis of p-p65 and p -IRF3 in indicated THP1 cells following stimulation with 3′3'-cGAMP/LPS for 6 h. (B and C) In parallel, the activity of IRF or NF-κB was assayed by luciferase reporter gene assay (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (D and E) Analysis of ACOD1 mRNA in indicated THP1 or RAW264.7 cells following stimulation with 3′3′-cGAMP/LPS for 3 and 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (F) Heatmap of kinase phosphorylation changes in indicated THP1 cells following 3′3'-cGAMP/LPS stimulation for 6 h. (G) IP analysis of IRF3-JUN protein complex in THP1 cells following stimulation with 3′3′-cGAMP/LPS for 6 h. (H and I) ChIP-qPCR analysis of the binding of IRF3 and JUN on the promoter of ACOD1 in THP1 cells following stimulation with 3′3′-cGAMP/LPS for 3 and 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; t test). (J) Western blot analysis of ACOD1 in indicated THP1 cells following stimulation with 3′3′-cGAMP/LPS for 6 h.

Article Snippet: STING1-V155M THP1 , InvivoGen , thpd-m155.

Techniques: Western Blot, Activity Assay, Luciferase, Reporter Gene Assay, Binding Assay

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: STING1-mediated itaconate production promotes endotoxemia (A) Analysis of animal survival in indicated mice with or without repeated intraperitoneal administration of 4OI (50 mg/kg) at 2, 24, 48, and 72 h after LPS (15 mg/kg) treatment (n = 15 mice/group; Kaplan-Meier survival analysis). (B–H) In parallel, indicated plasma biomarkers (C-F), as well as Acod1 mRNA (G) and itaconate (H) in peritoneal macrophages, were assayed (Data are presented as mean ± SD; n = 5 mice/group; one-way ANOVA with Tukey’s multiple comparisons test). (I–K) Peritoneal macrophages were treated with 4OI (1 mM) for 24 and 48 h, and cell death and the release of HMGB1 and SQSTM1 were assayed (Data are presented as mean ± SD; n = 5 biologically independent samples; one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: STING1-V155M THP1 , InvivoGen , thpd-m155.

Techniques:

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: STING1-mediated itaconate production promotes polymicrobial sepsis (A) Analysis of animal survival in indicated mice with or without repeated intraperitoneal administration of 4OI (50 mg/kg) at 2, 24, 48, and 72 h following CLP procedure (n = 15 mice/group; Kaplan-Meier survival analysis). (B–H) In parallel, indicated plasma biomarkers (C-F), as well as Acod1 mRNA (G) and itaconate (H) in peritoneal macrophages, were assayed (Data are presented as mean ± SD; n = 5 mice/group; one-way ANOVA with Tukey’s multiple comparisons test). (I) Schematic summary of the role of STING1 in driving ACOD1 expression and itaconate production for sepsis.

Article Snippet: STING1-V155M THP1 , InvivoGen , thpd-m155.

Techniques: Expressing

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet:

Article Snippet: STING1-V155M THP1 , InvivoGen , thpd-m155.

Techniques: Recombinant, Control, Lysis, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Magnetic Beads, Chromatin Immunoprecipitation, Purification, CCK-8 Assay, shRNA, Software, Western Blot

Journal: Cell reports

Article Title: FEZ1 phosphorylation regulates HSPA8 localization and interferon-stimulated gene expression

doi: 10.1016/j.celrep.2022.110396

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: THP1-Dual (thpd-nfis) and THP1-Dual KO-STING (thpd-kostg) cell lines stably expressing inducible NF-κB-SEAP and IRF-Lucia luciferase reporters were purchased from InvivoGen.

Techniques: Virus, Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Extraction, Protease Inhibitor, Software, 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 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: 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