Journal: Nature Communications
Article Title: Clustering of NLRP3 induced by membrane or protein scaffolds promotes inflammasome assembly
doi: 10.1038/s41467-025-60277-4
Figure Lengend Snippet: a Conserved basic segments within the linker and FISNA region of NLRP3 homologs. b Immunoblot of non-localized and organelle-tethered NLRP3-YFP with respective NLRP3 mutants K127A/K128A/K129A/K130A (4xA). c – h Confocal images of NLRP3-KO iBMDMs stably transduced with organelle-tethered NLRP3-YFP (yellow) variants 12 h post priming with or without nigericin stimulation (Nig), stained for nuclei (Hoechst 33342, cyan) and plasma membrane (Cholera Toxin Subunit B, CTB, magenta). Scale bar, 10 µm. Following priming, 10 µM nigericin, 0.2 mg/ml SiO 2 or 20 ug/ml imiquimod (Imq) were added to cells, respectively, as indicated in the respective graph columns with legends above. Measurements of IL-1β were normalized to each respective non-mutated, organelle-enriched NLRP3-YFP: WT ( c ), Cb5 ( d ), TGN38 ( e ), HRas ( f ), Rheb ( g ) or Omp25 ( h ) treated with canonical stimuli: nigericin, SiO 2 or imiquimod, respectively. EV, empty vector. Data represents the mean ± SEM of three independent experiments, except for TGN38, treated with SiO 2 in ( e ), which is the mean ± SEM of two independent experiments. The average value calculated from technical replicates within an individual experiment is presented as a single data point. P values were calculated using two-tailed unpaired t -test with Welch’s correction ( c – h ). Western blots ( b ) and microscopic images ( c – h ) are representative of three independent experiments. Panel ( a ) created in Biorender. Hafner Bratkovic, I. (2025) https://BioRender.com/99kdc99 .
Article Snippet: Localization sequences were amplified from plasmids encoding Hras (a gift from D. Sabatini, Addgene 26637 ), Tmem192 (a gift from D. Sabatini, Addgene 102930 ), Rheb (a gift from D. Sabatini, Addgene 26634 ), TGN38 (a gift from J. Lippincott-Schwartz, Addgene 128148), GOLGB1 (a gift from D. Gadella, Addgene 67903 ), Omp25 (a gift from D. Sabatini, Addgene 26638 ), PEX3 (a gift from J. Boehm & W. Hahn & D. Root, Addgene 81779 ), PEX26 (a gift from D. Sabatini, Addgene 139054 ) and PACT (a gift from C. Norden, Addgene 105954 ).
Techniques: Western Blot, Stable Transfection, Transduction, Staining, Clinical Proteomics, Membrane, Plasmid Preparation, Two Tailed Test
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![Figure 4. The ORF3a-∆YxxΦ is not expressed at the cell-plasma membrane. HEK293 cells were transfected with the empty pcDNA.3.1(+) vector or a vector expressing the SARS-CoV-2 HA-ORF3a- [∆YxxΦ] protein section as in Figure 3. (A). Cells transfected with vectors expressing HA-ORF3a- [∆YxxΦ] and a vector expressing a marker for the rough endoplasmic reticulum (RER; ERmoxGFP) and immunostained with an anti-HA antibody. (B). Cells transfected with a vector expressing HA-ORF3a-[∆YxxΦ] and immunostained with antibodies against ERGIC-53 and HA. (C). Cells trans- fected with vectors expressing HA-ORF3a-[∆YxxΦ] and mNeonGreen-Giantin and immunostained with an anti-HA antibody. (D). Cells transfected with the vector expressing HA-ORF3a-[∆YxxΦ] and immunostained with antibodies against Golgin 97 and HA. (E). Cells transfected with vectors expressing HA-ORF3a-[∆YxxΦ] and TGN38 EGFP and immunostained with an anti-HA antibody. (F). Cells transfected with the vector expressing HA-ORF3a-[∆YxxΦ] and 4xmts-mNeonGreen and immunostained with antibodies against HA.](https://doi-unpaywalled-images-cdn.bioz.com/8064/10__3390_slash_v17040522/10__3390_slash_v17040522____page12_image1.jpg)
![Figure 5. Cell expression of ORF3a mutants with one tyrosine-based motif intact. COS-7 cells were co-transfected with vectors expressing HA-ORF3a, HA-ORF3a-[Y160], HA-ORF3a-[Y211], or HA-ORF3a-[Y233] and a vector expressing ER-moxEGFP or TGN38-EGFP. At 48 h post-transfection, cells were fixed, permeabilized, and blocked. Cells were reacted with a mouse monoclonal antibody against the HA-tag overnight, and washed and reacted with an appropriate secondary antibody tagged with Alexa Fluor 594 (for HA) for 1 h. Cells were washed and counter-stained with DAPI (1 µg/mL) for 5 min. Cells were viewed using a Leica TC8 confocal microscope as described in the Section 2. At least 50 cells were examined for surface expression and co-localization with ERmoxGFP or TGN38 EGFP. (A). Cells transfected with a vector expressing HA-ORF3a-[Y160] and ERmoxGFP and immunostained with antibodies against the HA-tag. (B). Cells transfected with a vector expressing HA-ORF3a-[Y160] and immunostained with antibodies against the HA-tag and ERGIC-53. (C). Cells transfected with a vectors expressing HA-ORF3a-[Y160] and TGN38 EGFP and immunostained with an antibody against the HA-tag. (D). Cells transfected with vectors expressing HA-ORF3a-[Y211] and ERmoxGFP and immunostained with antibodies against the HA-tag. (E). Cells transfected with a vector expressing HA-ORF3a-[Y160] and immunostained with antibodies against the HA-tag and ERGIC-53. (F). Cells transfected with a vector expressing HA-ORF3a-Y211 and TGN38 EGFP and immunostained with antibodies against the HA-tag. (G). Cells transfected with](https://doi-unpaywalled-images-cdn.bioz.com/8064/10__3390_slash_v17040522/10__3390_slash_v17040522____page13_image1.jpg)
![Figure 6. The cell expression of ORF3a mutants with two intact tyrosine motifs intact. COS-7 cells were co-transfected with vectors expressing HA-ORF3a-[Y160,211] (A–C), HA-ORF3a-[Y160,233] (D–F), or HA-ORF3a-[Y211,233] (G–I) and ERmoxGFP (A,D,G) or TGN38-GFP (C,F,I). Cells were also transfected with the ORF3a mutants above and immunostained for ERGIC-53 (B,E,H). At 48 h post- transfection, cells were fixed, permeabilized, and blocked. Cells were reacted with a mouse monoclonal antibody against the HA-tag overnight, washed, and reacted with an appropriate secondary antibody tagged with Alexa Fluor 594 for HA for 1 h. Cells were washed and counter-stained with DAPI (1 µg/mL) for 5 min. Cells were viewed using a Leica TC8 confocal microscope as described in the Section 2. At least 50 cells were examined for expression and co-localization. (A–C). Cells transfected with vectors expressing HA-ORF3a-[Y160,211] and](https://doi-unpaywalled-images-cdn.bioz.com/8064/10__3390_slash_v17040522/10__3390_slash_v17040522____page14_image1.jpg)