Journal: bioRxiv

Article Title: Full-length NLRP3 forms oligomeric cages to mediate NLRP3 sensing and activation

doi: 10.1101/2021.09.12.459968

Figure Lengend Snippet: a, b , Confocal imaging of WT ( a ) and NLRP3 -/- ( b ) iBMDMs primed with LPS (top) or also treated with 20 µM nigericin for 1 h (bottom) by IF with goat anti-NLRP3 (magenta) and rabbit anti-TGN38 (green) antibodies, and DNA (Hoechst dye, blue). NLRP3 inflammasome specks are labelled with arrowheads. TGN dispersion requires NLRP3. c , Confocal imaging of NLRP3 -/- iBMDMs reconstituted with WT human mScarlet-NLRP3 primed with LPS (top) or also treated with 20 µM nigericin for 1 h (bottom) for TGN38 (IF, green), NLRP3 (mScarlet, magenta) and DNA (Hoechst dye, blue). These reconstituted cells induced TGN dispersion upon nigericin stimulation identical to WT iBMDMs. d , Confocal imaging of NLRP3 -/- iBMDMs reconstituted with WT human mScarlet-NLRP3 treated with LPS and MCC950 (top) or pre-treated with MCC950 and treated with 20 μM nigericin for 1 h (bottom) for TGN38 (IF, green), NLRP3 (mScarlet, magenta) and DNA (Hoechst dye, blue). e-i , Confocal imaging of NLRP3 -/- iBMDMs reconstituted with WT ( e ) or double ring cage disrupting mutants of human mScarlet-NLRP3 (LRRm2, 3, 5 and ΔPYD) ( f-i ) primed with LPS (top) or also treated with 20 µM nigericin for 1 h (bottom) for 58K Golgi protein (IF, green), NLRP3 (mScarlet, magenta) and DNA (Hoechst dye, blue). Disruption of the double ring cage abolished TGN dispersion upon nigericin treatment. j, k , Confocal imaging of NLRP3 -/- iBMDMs reconstituted with NLRP3 linker-mutant ( j ) or OSBP-linker mutant ( k ) primed with LPS (top) or also treated with 20 µM nigericin for 1 h (bottom) for 58K Golgi protein (IF, green), NLRP3 (mScarlet, magenta) and DNA (Hoechst dye, blue). Targeting the linker mutant to the membrane rescued TGN dispersion. All images are maximum intensity Z projections with scale bars of 10 μm. l , Quantification of TGN dispersion based on confocal imaging for WT iBMDMs, NLRP3 -/- iBMDMs, and NLRP3 -/- iBMDMs reconstituted with WT or mutant human mScarlet-NLRP3, with or without the Golgi-binding OSBP domain. Disruption of the NLRP3 double ring cage abolished TGN dispersion upon nigericin treatment. m , Cell death indicated by LDH release for WT iBMDMs, NLRP3 -/- iBMDMs, and NLRP3 -/- iBMDMs reconstituted with WT or mutant NLRP3, with or without the Golgi-binding OSBP domain. The level of LDH release is shown as a fold change between LPS-primed cells treated or not with nigericin. Data are presented as mean ± s.d., n=3. Double ring cage disrupting mutations abolished NLRP3-mediated cell death.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-NLRP3 (1:250, Abcam, Cat. no: ab272702), goat polyclonal anti-NLRP3 (1:100, Abcam, Cat. no: 4207), rabbit polyclonal anti-TGN38 (1:500, Novus Biologicals, Cat. no: NBP1-03495), rabbit polyclonal anti-TGN46 (1:500, Abcam, Cat. no: 50595), mouse monoclonal anti-58K Golgi (1:500, Novus Biologicals, Cat. no: NB600-412), sheep polyclonal anti-TGN38 (1:500, Novus Biologicals, Cat. no: NBP1-20263), rabbit polyclonal anti-ASC (1:1000, Cell Signaling, Cat. no: 67824) and mouse monoclonal anti-γ-tubulin (1:500, Abcam, Cat. no: ab11316).

Techniques: Imaging, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Full-length NLRP3 forms oligomeric cages to mediate NLRP3 sensing and activation

doi: 10.1101/2021.09.12.459968

Figure Lengend Snippet: a , Confocal imaging of WT (top) and NLRP3 -/- iBMDMs (bottom) primed with LPS and also treated with 20 μM nigericin for 1 h for γ-tubulin (IF, cyan), ASC (IF, yellow), and DNA (Hoechst dye, blue). NLRP3 inflammasome specks are labelled with arrowheads. b, c , Confocal imaging of WT ( b ) and NLRP3 -/- iBMDMs ( c ) primed with LPS (top) or also treated with 20 μM nigericin for 1 h (bottom) by IF for NLRP3 (rabbit anti-NLRP3 antibody, magenta), 58K Golgi protein (green) and DNA (Hoechst dye, blue). NLRP3 inflammasome specks are labelled with arrowheads. d , Confocal imaging of WT iBMDMs primed with LPS (top) or also treated with 20 μM nigericin for 1 h (bottom) by IF for ASC (yellow), 58K Golgi protein (green) and DNA (Hoechst dye, blue). NLRP3 inflammasome specks are labelled with arrowheads. e, f , Channel controls for immunofluorescence in ( b-c ) and . WT iBMDMs were visualized by IF for NLRP3 (goat or rabbit anti-NLRP3 antibody, magenta), TGN38 (rabbit anti-TGN38 antibody, green) or 58K Golgi protein (green) in LPS-primed cells. NLRP3 was detected by IF with Alexa488-labeled secondary antibody, TGN38 by IF with Alexa647-labeled secondary antibody, and DNA by Hoechst dye ( e ). NLRP3 was detected by IF with Alexa647-labeled secondary antibody, the 58K Golgi protein by IF with Alexa488-labeled secondary antibody, and DNA by Hoechst dye ( f ). g , Isotype controls for the immunofluorescence. LPS-primed WT iBMDMs were incubated with goat IgG, rabbit IgG or no primary antibody and visualized by IF with anti-goat Alexa488- or anti-rabbit Alexa647-labeled antibodies, respectively. h , Confocal imaging of WT iBMDMs primed with LPS (top) or also pre-treated with MCC950 and treated with 20 μM nigericin for 1 h (bottom) for TGN38 (IF, green), NLRP3 (mScarlet, magenta) and DNA (Hoechst dye, blue). Z projections with scale bars of 10 μm. i , WBs of the whole cell lysates from LPS-, MCC950+nigericin- and nigericin-treated WT iBMDMs, NLRP3 -/- iBMDMs, or NLRP3 -/- reconstituted with human WT mScarlet-NLRP3. FLAG-mScarlet-NLRP3, NLRP3 and cleaved caspase-1 were visualized with corresponding antibodies. MCC950 inhibited caspase-1 processing. j , Cell death indicated by LDH release for WT iBMDMs, NLRP3 -/- iBMDMs, and NLRP3 -/- iBMDMs reconstituted with WT mScarlet-NLRP3 treated with nigericin only or pretreated with MCC950. The level of LDH release is shown as a fold change between LPS-primed cells treated or not with nigericin. Data are presented as mean ± s.d., n=3. MCC950 abolished NLRP3-mediated cell death. k , Confocal imaging of NLRP3 -/- iBMDMs reconstituted with OSBP-fused double ring cage disrupting mutant LRRm2 of human mScarlet-NLRP3 primed with LPS (top) or also treated with 20 μM nigericin for 1 h (bottom) for 58K Golgi protein (IF, green), NLRP3 (mScarlet, magenta), ASC (yellow), and DNA (Hoechst dye, blue). Disruption of the double ring cage abolished TGN dispersion upon nigericin treatment even when this NLRP3 mutant was targeted to TGN by the OSBP domain. All images are maximum intensity Z projections with scale bars of 10 μm.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-NLRP3 (1:250, Abcam, Cat. no: ab272702), goat polyclonal anti-NLRP3 (1:100, Abcam, Cat. no: 4207), rabbit polyclonal anti-TGN38 (1:500, Novus Biologicals, Cat. no: NBP1-03495), rabbit polyclonal anti-TGN46 (1:500, Abcam, Cat. no: 50595), mouse monoclonal anti-58K Golgi (1:500, Novus Biologicals, Cat. no: NB600-412), sheep polyclonal anti-TGN38 (1:500, Novus Biologicals, Cat. no: NBP1-20263), rabbit polyclonal anti-ASC (1:1000, Cell Signaling, Cat. no: 67824) and mouse monoclonal anti-γ-tubulin (1:500, Abcam, Cat. no: ab11316).

Techniques: Imaging, Immunofluorescence, Labeling, Incubation, Mutagenesis