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: bioRxiv

Article Title: Bifidobacterium pseudocatenulatum extracellular vesicles promote Ly6G+ granulocyte infiltration to inhibit melanoma tumour progression

doi: 10.64898/2026.06.08.731027

Figure Lengend Snippet: (A) Bone marrow cells were incubated ex vivo with PBS or BEVs (10 9 particles/well) for 5 h. CD11b+Ly6G+ cells among CD45+ cells were quantified by flow cytometry; representative contour plots shown. (B) ICAM-1 expression on Ly6G+ cells after BEV exposure; representative plots and quantification shown. (C-D) In vivo bone marrow CD11b+ and Ly6G+ populations among CD45+ cells in non-tumour-bearing and tumour-bearing mice following PBS or BEV administration. (E) KC/CXCL1 levels in mouse splenocyte supernatants following PBS, BEV or LPS stimulation. (F) IL-8 levels in THP-1 macrophage and THP1-Blue supernatants following PBS, BEV or LPS stimulation. Statistics: (A-B) paired two-tailed t-tests; other statistical tests as indicated in Methods. Bars = mean ± SEM.

Article Snippet: THP-1 monocytes (ATCC, TIB-202), THP1-Blue NF-kB reporter monocytes and HEK-Blue hTLR2/hTLR4 reporter cells (InvivoGen) were cultured at 37°C/5% CO 2 in RPMI-1640 (Gibco) containing L-glutamine (2 mM) and HEPES (25 mM), supplemented with heat-inactivated FBS.

Techniques: Incubation, Ex Vivo, Flow Cytometry, Expressing, In Vivo, Two Tailed Test, FACS

Journal: Frontiers in Immunology

Article Title: Isolation of a nanobody specific to the PstS-1 protein and evaluation of its immunoreactivity with structural components of Mycobacterium tuberculosis granuloma

doi: 10.3389/fimmu.2025.1684904

Figure Lengend Snippet: C8Nb recognizes PstS-1 protein adhered on the surface of either mycobacteria or macrophages. (A) When single cell suspensions of bacteria were probed with 125 I-C8Nb, only Mtb and BCG pellets demonstrated tracer binding. (B) Immunoreactivity of 125 I-C8Nb was confirmed with single cell suspensions of Mtb LAM and Mtb Beijing strains. (C) Protein mixtures prepared from bacterial lysates or rPstS-1 protein were adhered on formaldehyde fixed RAW 264.7 macrophages and then cells were incubated with 125 I-C8Nb. Tracer showed binding with the cells which were treated with either rPstS-1 protein or protein mixtures prepared from Mtb or BCG lysates. (D) In similar binding studies on THP-1 macrophages adhered with rPstS-1 protein, WT and TLR4 KO cells demonstrated comparable tracer binding, while tracer binding with TLR2 KO cells was low and comparable with WT cells treated with BSA. In the case of MR, spiking Mtb protein mixture with mannose (1mg/ml) did not affect tracer binding. Data suggested that the epitope on PstS-1 protein bound to only TLR2 is accessible to C8Nb for binding, while MR and TLR4 are not playing role in binding. In all assays (A-D) , specific binding of 125 I-C8Nb tracer (T) was assessed by including spiked tracer (ST). (E) In fluorescence microscopy, THP-1 cells were sequentially incubated with rPstS-1 protein, C8Nb and rabbit anti-camelid V H H cocktail-iFluor555. Analysis of acquired images suggested that TLR2 is involved in binding of C8Nb with PstS-1 protein treated THP-1 macrophages. Scale bars: 10μm. (A-D) all values are mean ± SD, and derived from more than three independent experiments. Significance was calculated by one-way ANOVA followed by Dunnett’s multiple comparisons or Bonferroni’s post hoc test; ***P <0.001.

Article Snippet: TLR-2 and TLR-4 knockout (KO) THP-1 cells (Invivogen) were cultured and maintained in RPMI 1640 medium under selection pressure of blasticidin (10μg/ml) and zeocin (100μg/ml).

Techniques: Single Cell, Bacteria, Binding Assay, Incubation, Fluorescence, Microscopy, Analysis, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Discovery of potent and selective inhibitors of human NLRP3 with a novel mechanism of action

doi: 10.1084/jem.20242403

Figure Lengend Snippet: BAL-0028 specifically inhibits NLRP3 inflammasome formation. (A) Western blot for caspase-1 and IL-1β cleavage and NLRP3 expression from PMA-differentiated THP-1 cells stimulated with LPS and nigericin in the presence of BAL-0028 or MCC950 (both 500 nM). (B) Comparison of BAL-0028 and MCC950 effects on ASC speck formation assessed by fluorescence microscopy in PMA-differentiated THP-1 ASC-GFP cells stimulated with LPS and nigericin. (C) Comparison of BAL-0028 and MCC950 effects on ASC speck formation assessed by flow cytometry in HEK293T ASC-BFP cells transfected with human NLRP3 and stimulated with nigericin. (D and E) Effect of BAL-0028 and MCC950 on ASC speck formation assessed by fluorescence microscopy using an anti-ASC antibody in iMacs. (F and G) Effects of BAL-0028, MCC950, and VX-765 on IL-1β release from PMA-differentiated THP-1 cells stimulated with (F) LPS and transfected with poly(dA:dT) or (G) protective antigen (PA) and Lfn-needle protein. (H) Effects of BAL-0028, MCC950, and VX-765 on IL-18 release from human keratinocytes stimulated with talabostat. (A) Representative blots from N = 2 independent experiments. (B) Average ± SEM % ASC-GFP speck-positive cells from N = 2 independent experiments performed in triplicate. (C) Average ± SEM change in nigericin-induced ASC specks normalized to cells without compound treatment from N = 3–4 independent experiments. (D and F–H) Graph symbols show average values relative to vehicle control from independent experiments performed in triplicate (indicated by different symbols) ± SEM. N = 2 (D, G, and H) and N = 3 (F). (E) Representative images from D, scale bar is 100 μM. Source data are available for this figure: .

Article Snippet: THP-1-ASC-GFP (InvivoGen) were maintained in growth media containing RPMI-1640 + GlutaMAX (Gibco) with 10% FBS (Corning) and 1% pen/strep (Caisson) at 37°C in a 5% CO 2 atmosphere.

Techniques: Western Blot, Expressing, Comparison, Fluorescence, Microscopy, Flow Cytometry, Transfection, Control, Fluorescence Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Discovery of potent and selective inhibitors of human NLRP3 with a novel mechanism of action

doi: 10.1084/jem.20242403

Figure Lengend Snippet: BAL-0598 inhibits the activation of human and monkey NLRP3 and is a non-covalent inhibitor . Related to and . (A) BAL-0598 in IL-1β release assay from PMA-differentiated THP-1 cells stimulated with LPS and MSU. (B) BAL-0598 effect on ASC speck formation assessed by fluorescence microscopy in PMA-differentiated THP-1 ASC-GFP cells stimulated with LPS and nigericin. (C and D) Comparison of BAL-0598, BAL-0028, and MCC950 in an LDH release assay in primary peritoneal macrophages isolated from (C) WT 129S6 or (D) 129S6 mouse promoter- NLRP3 mice stimulated with LPS and ATP. Graph symbols show average LDH values ± SEM from N = 2 independent experiments performed in duplicate. (E and F) BAL-0598 in IL-1β release assays from cells stimulated with LPS and nigericin. African green monkey CD14 + monocytes (E) and PBMCs (F). (A, B, E, and F) Graph symbols show average values relative to vehicle control ± SEM (A, N = 2) or SD (B, E, and F, N = 1) from independent experiments performed in duplicate (E) or triplicate (A, B, and F); IC 50 curve was fitted by nonlinear regression analysis. (G) Comparison of BAL-0028, BAL-0598, and MCC950 in an IL-1β release assay from PMA-differentiated THP-1 cells stimulated with LPS and nigericin. Cells were treated with compounds before nigericin stimulation, and compounds were left on or were washed out for 1 min before nigericin addition. The graph shows average IL-1β values ± SD from one experiment performed in triplicate. (H) Schematic illustration of U937 NLRP3 and NLRP3-AID mutant cell model.

Article Snippet: THP-1-ASC-GFP (InvivoGen) were maintained in growth media containing RPMI-1640 + GlutaMAX (Gibco) with 10% FBS (Corning) and 1% pen/strep (Caisson) at 37°C in a 5% CO 2 atmosphere.

Techniques: Activation Assay, Release Assay, Fluorescence, Microscopy, Comparison, Lactate Dehydrogenase Assay, Isolation, Control, Analysis, Mutagenesis