human nlrp3 mcherry constructs  (Addgene inc)

Journal: bioRxiv

Article Title: Sterile- or pathogen-induced endolysosomal damage activate the NLRP6 inflammasome in human intestinal epithelial cells

doi: 10.1101/2025.01.23.634286

Figure Lengend Snippet: A. Schematic representation of human NLRP6 and NLRP3 showing their domain structures and the respective polybasic regions within the FISNA of mouse and human NLRP3 and 6 aligned. B. Flow cytometry-based quantification of ASC speck formation in GFP-ASC tg HEK293T cells expressing hNLRP6-mCherry or hNLRP3-mCherry infected with WT or τ< hly L. monocytogenes EGD for 6h, treated with nigericin for 1h or with imiquimod for 6h in presence or absence of 60 mM extracellular KCl. C. Flow cytometry based quantification of ASC speck formation in GFP-ASC tg HEK293T cells, expressing the indicated NLRP6 proteins, infected with WT or τ< hly L. monocytogenes EGD for 6h D. Flow cytometry based quantification of ASC speck formation in GFP-ASC tg HEK293T cells expressing either NLRP6-WT, NLRP3-WT or the indicated chimeric NLRP6-3 proteins with mCherry tags, infected with WT or τ< hly L. monocytogenes EGD for 6h, treated with nigericin for 1h or with imiquimod for 6h. E. Flow cytometry based quantification of ASC speck formation in GFP-ASC tg HEK293T cells, expressing NLRP6 τιLRR -mCherry, infected with WT or τ< hly L. monocytogenes EGD for 6h. F. Flow cytometry based quantification of ASC speck formation in GFP-ASC tg HEK293T cells, expressing NLRP6 WA/WB , infected with WT or τ< hly L. monocytogenes EGD for 6h. Graphs show mean ± SD from three pooled experiments. ns = non-significant, **P < 0.01; ***P < 0.001, ****P < 0.0001 (two-way ANOVA with either Tukey’s multiple comparisons test (B), Dunett’s multiple comparisons test (C,E) or Uncorrected Fisher’s LSD (F)).

Article Snippet: Human NLRP3-mCherry constructs are derived from a pEGFP-C2-NLRP3 plasmid (addgene #73955), while human NLRP6-mCherry constructs were derived from the plasmid mentioned above.

Techniques: Flow Cytometry, Expressing, Infection

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Indole-3-Aldehyde alleviates lung inflammation in COPD through activating Aryl Hydrocarbon Receptor to inhibit HDACs/NF-κB/NLRP3 signaling pathways

doi: 10.1007/s00109-024-02506-9

Figure Lengend Snippet: List of Antibodies used in this study

Article Snippet: For NLRP3 overexpression, cells were transfected with 1 μg of pcDNA3.1-Flag plasmid (addgene) or of NLRP3 construct (addgene) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.

Techniques:

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Indole-3-Aldehyde alleviates lung inflammation in COPD through activating Aryl Hydrocarbon Receptor to inhibit HDACs/NF-κB/NLRP3 signaling pathways

doi: 10.1007/s00109-024-02506-9

Figure Lengend Snippet: Specific primers for RT- PCR

Article Snippet: For NLRP3 overexpression, cells were transfected with 1 μg of pcDNA3.1-Flag plasmid (addgene) or of NLRP3 construct (addgene) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.

Techniques:

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Indole-3-Aldehyde alleviates lung inflammation in COPD through activating Aryl Hydrocarbon Receptor to inhibit HDACs/NF-κB/NLRP3 signaling pathways

doi: 10.1007/s00109-024-02506-9

Figure Lengend Snippet: I3A treatment inhibited the activated NLRP3 signaling pathway by CSE stimulation in MH-S cells. a . The protein expression levels of NLRP3, ASC, Caspase-1, and IL-1β were detected by WB, with GAPDH serving as an internal reference protein. The relative band densities of NLRP3, ASC, Caspase-1, and IL-1β are shown in Fig. 4b, c, d, and e, respectively. CTRL, control; a minus sign ( −), absent;(* p < 0.05,** p < 0.01, *** p < 0.001 in one-way ANOVA with Tukey’s multiple comparison)

Article Snippet: For NLRP3 overexpression, cells were transfected with 1 μg of pcDNA3.1-Flag plasmid (addgene) or of NLRP3 construct (addgene) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.

Techniques: Expressing, Control, Comparison

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Indole-3-Aldehyde alleviates lung inflammation in COPD through activating Aryl Hydrocarbon Receptor to inhibit HDACs/NF-κB/NLRP3 signaling pathways

doi: 10.1007/s00109-024-02506-9

Figure Lengend Snippet: Inhibition of HDAC5/6 activity inactivated the NF-κB/NLRP3 pathways. a . The mRNA levels of HDAC1-11 were detected by RT-PCR, with GAPDH serving as an internal control. b . Efficiencies of siRNA-mediated knockdown of HDACs (HDAC5, HDAC6 or HDAC5/6) in MH-S cells were determined via RT-PCR. c . The levels of inflammatory cytokines in the cell supernatant in the different groups were examined by ELISA. d . Treatment with siHDAC5/6 inhibited the NF-κB and NLRP3 signaling pathways, as determined by Western blot analysis of the relevant proteins. e . The band intensities of proteins shown in Fig. 5d were analyzed. CTRL, control; NC, negative control; ns, non-significance; a minus sign ( −), absent; (* p < 0.05, ** p < 0.01, *** p < 0.001 in one-way ANOVA with Tukey’s multiple comparison)

Article Snippet: For NLRP3 overexpression, cells were transfected with 1 μg of pcDNA3.1-Flag plasmid (addgene) or of NLRP3 construct (addgene) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.

Techniques: Inhibition, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Control, Knockdown, Enzyme-linked Immunosorbent Assay, Protein-Protein interactions, Western Blot, Negative Control, Comparison

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Indole-3-Aldehyde alleviates lung inflammation in COPD through activating Aryl Hydrocarbon Receptor to inhibit HDACs/NF-κB/NLRP3 signaling pathways

doi: 10.1007/s00109-024-02506-9

Figure Lengend Snippet: Knocking down AHR abolished the anti-inflammatory effect of I3A. a . The protein levels of HDAC5/6 were detected by WB. b . Relative band densities of HDAC5/6 shown in Fig. 6a were analyzed. c . Efficiency of siRNA-mediated knockdown of AHR in MH-S cells was determined by RT-PCR. d . The mRNA levels of AHR in MH-S cells stimulated by cigarette smoke (CS) or I3A treatment were assessed using RT-PCR. e . The levels of inflammatory cytokines in the cell supernatant in the different groups were examined by ELISA. f . Effects of down-regulation of AHR on MMPs, NF-κB/NLRP3 signaling pathways were performed by WB. g . Relative band intensities of proteins shown in Fig. 6e were analyzed. CTRL, control; NC, negative control; ns, non-significance; a minus sign ( −), absent; (* p < 0.05, ** p < 0.01, *** p < 0.001 in one-way ANOVA with Tukey’s multiple comparison)

Article Snippet: For NLRP3 overexpression, cells were transfected with 1 μg of pcDNA3.1-Flag plasmid (addgene) or of NLRP3 construct (addgene) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Protein-Protein interactions, Control, Negative Control, Comparison