Journal: bioRxiv

Article Title: NLRP3 activators disrupt the endocytic AP2 complex and plasma membrane signaling

doi: 10.64898/2026.02.24.707400

Figure Lengend Snippet: (A) IL-1β release of LPS-primed wt Balb/c iBMDMs treated with Dyngo-4a, Dynasore (40/ 80/ 160 µM), ATP (2 mM) or Nigericin (2 µM) for 90 min; n = 3. (B) IL-1β release of LPS-primed C57Bl6 BMDMs stimulated with Nigericin (2 µM), Dyngo-4a (80 µM) or Dynasore (160 µM) in presence of excessive KCl (0-80 mM) or the Caspase-1 inhibitor VX-765 (10 µM); n = 3 (C) Quantification of intracellular K + via ion-selective electrode measurement. MCC-950 indicates Nlrp3 inhibitor treatment. (D) Representative Western blot for cleaved (p20) and uncleaved (p45) caspase-1 from primary C57Bl6 BMDM supernatants; n = 3 (E) As in C, but in presence of increasing concentrations of KCl; n = 3. (F) Representative Western blot for mature and pro-IL-1β in presence or absence of excess 80 mM KCl in wt Balb/C iBMDMs, NLRP3- or Caspase-1/11 deficient iBMDMs, treated with 80 µM Dynasore, 10 µM Nigericin, 60 µg/ml R837 (left) or 20/ 40/ 80 µM Dynasore or Dyngo-4a (right); n = 2. (G) Relative AlexaFlour-647 labelled transferrin (Tfn, left) or AlexaFlour-594 labelled Choleratoxin-B (CtxB, right) uptake in ASC -/- iBMDMs in the presence of Dyngo-4a or Dynasore; n = 3 (H) Enrichment analysis for GOCC terms and Uniprot keywords using Fisher exact test on proteins significantly (p < 0.05; FDR < 0.02) changing localization in ASC -/- iBMDMs in presence of Dyngo-4a (80 µM), Dynasore (160 µM) or Nigericin (2 µM) relative to LPS-primed control. (I) Profile plots showing relative AP2-complex subunit (AP2a1, AP2a2, AP2b1, AP2m1, AP2s1) distribution across fractions. (J) 3D-Principal-component-analysis showing transition of AP2-complex subunits (grey-LPS, black-Nigericin, movement indicated by lines) upon 80 µM Dyngo-4a (left) or 160 µM Dynasore (right) treatment with annotations for endosomal (red), Golgi- (orange) or plasma membrane proteins (violet). (K) Representative confocal microscopy images of LPS-primed ASC -/- iBMDMs stably expressing functional AP2a1-eGFP-AP2b1 fusion protein and transiently expressing NLRP3-tagRFP, co-stained with DAPI. Statistics indicate significance by student’s two-tailed t-test (A, B, C, F, G).

Article Snippet: For spatial proteomics experiments, per replicate, approximately 100×10 6 Casp1/11 -/- or Asc -/- SILAC-heavy or -light labelled iBMDMs were primed with 1 μg/ml LPS-EB or LPS-EB Ultrapure from E. coli 0111:B4 (InvivoGen, #tlrl-eblps or #tlrl-3pelps) in CM for 4 h at 37°C, 5% CO 2 , followed by stimulation in serum free conditions with either 10 μM Nigericin (NI; Thermo Fisher Scientific, #N1495), 60 μg/ml R837/Imiquimod (InvivoGen, #tlrl-imqs-1), 10 μM monensin (Merck, #M5273), Mdivi-1 (Selleckchem, #S7162), K + -free Hank’s balanced salt solution (HBSS), 160 μM Dynasore (DS; Selleckchem, #S8047), 80 μM Dyngo-4a (DN; Selleckchem, #S7163), or serum-free DMEM as control.

Techniques: Western Blot, Control, Clinical Proteomics, Membrane, Confocal Microscopy, Stable Transfection, Expressing, Functional Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: NLRP3 activators disrupt the endocytic AP2 complex and plasma membrane signaling

doi: 10.64898/2026.02.24.707400

Figure Lengend Snippet: (A) IL-1β release of LPS-primed human monocyte-derived macrophages stimulated with 160 µM Dynasore ± 10 µM MCC-950 or 80 µM Dyngo-4a. Statistics indicate significance by student’s two-tailed t-test. (B) Western blot analysis of alpha-adaptin (AP2a1) in LPS-primed ASC -/- iBMDMs after stimulation with 80 µM Dynasore, 40 µM Dyngo-4a or 2 µM Nigericin, using 10 µg protein per fraction per condition for individual centrifugation speeds after subcellular fractionation.(C) Western blot analysis of alpha-adaptin (AP2a1) in LPS-primed ASC -/- iBMDMs after stimulation with 2 µM Nigericin, 10 µM chloroquine, 5 µM bafilomycin-4a, 10 µM FCCP or 1 µM wortmannin using 10 µg protein per fraction per condition for individual centrifugation speeds after subcellular fractionation. (D) Reactive oxygen species analysis of CM-H2DCFDA-stained ASC -/- iBMDMs treated with 10 µM Nigericin, 60 µg/ml R837, 80 µM Dynasore or 40 µM Dyngo-4a for 60 min. Shown is mean fluorescent intensity. (E) IL-1β release of LPS-primed C57Bl5 BMDMs in medium or treated with a mitochondrial Dynamin DRP1-inhibitor Mdivi-1, 40 µM for 90 min. n = 3. (F) Conventional cytokine (TNFa or IL-6) and IL-1β release of LPS-primed iBMDMs treated with 80 µM Dynasore (DS), 40 µM Dyngo-4a (DN), 10 µM Nigericin (NI), 60 µg/ml R837 or 2 µM ATP or in combination with 10 µM MCC-950; n = 2. (G) Heatmap of mean fold change per fraction of proteins that change significantly comparing Dynasore to LPS, Nigericin to LPS and Dynasore to Dyngo-4a in 3 out of 3 replicates. (H) Median IL-1β release in time- and dose-titration of Dynasore treatment in wt or NLRP3-deficient iBMDMs, determined via ELISA. n = 3. (I) IL-1β release of LPS-primed iBMDMs which were depleted of NLRP1, AIM2, Caspase-1, Caspase-11 and ASC by siRNA stimulated with 160 µM Dynasore ± 10 µM MCC-950 or 80 µM or 2 µM Nigericin.

Article Snippet: For spatial proteomics experiments, per replicate, approximately 100×10 6 Casp1/11 -/- or Asc -/- SILAC-heavy or -light labelled iBMDMs were primed with 1 μg/ml LPS-EB or LPS-EB Ultrapure from E. coli 0111:B4 (InvivoGen, #tlrl-eblps or #tlrl-3pelps) in CM for 4 h at 37°C, 5% CO 2 , followed by stimulation in serum free conditions with either 10 μM Nigericin (NI; Thermo Fisher Scientific, #N1495), 60 μg/ml R837/Imiquimod (InvivoGen, #tlrl-imqs-1), 10 μM monensin (Merck, #M5273), Mdivi-1 (Selleckchem, #S7162), K + -free Hank’s balanced salt solution (HBSS), 160 μM Dynasore (DS; Selleckchem, #S8047), 80 μM Dyngo-4a (DN; Selleckchem, #S7163), or serum-free DMEM as control.

Techniques: Derivative Assay, Two Tailed Test, Western Blot, Centrifugation, Fractionation, Staining, Titration, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: NLRP3 activators disrupt the endocytic AP2 complex and plasma membrane signaling

doi: 10.64898/2026.02.24.707400

Figure Lengend Snippet: (A) Chemical structure of biotinylated Dynasore (left) and Dyngo-4a (right). (B) 1D-annotation enrichment of fold changes comparing iBMDMs treated with 100 µM bitoin-Dynasore (right) or 100 µM biotin-Dyngo-4a (left). (C) Heatmap of protein expression of knockdown targets in unprimed iBMDMs 48 h after siRNA-mediated knockdown. Shown are log2-transformed median LFQ values of n = 3; fold reduction for AP2a1 = 91.63%; NME1 = 83.65%; NME2 = 76.28%, NME3, 4 and AP2a1 were not detected after knockdown. (D) Viability (mean) of LPS-primed iBMDMs treated with the NLRC4 agonist BsaK alone, in combination with VX-765, MCC-950 or 10 µM Nigericin; n = 3. (E) Heatmap of individual regulated phosphosites according to the keywords in . (F) As F, but adjacent to .

Article Snippet: For spatial proteomics experiments, per replicate, approximately 100×10 6 Casp1/11 -/- or Asc -/- SILAC-heavy or -light labelled iBMDMs were primed with 1 μg/ml LPS-EB or LPS-EB Ultrapure from E. coli 0111:B4 (InvivoGen, #tlrl-eblps or #tlrl-3pelps) in CM for 4 h at 37°C, 5% CO 2 , followed by stimulation in serum free conditions with either 10 μM Nigericin (NI; Thermo Fisher Scientific, #N1495), 60 μg/ml R837/Imiquimod (InvivoGen, #tlrl-imqs-1), 10 μM monensin (Merck, #M5273), Mdivi-1 (Selleckchem, #S7162), K + -free Hank’s balanced salt solution (HBSS), 160 μM Dynasore (DS; Selleckchem, #S8047), 80 μM Dyngo-4a (DN; Selleckchem, #S7163), or serum-free DMEM as control.

Techniques: Expressing, Knockdown, Transformation Assay

Journal: bioRxiv

Article Title: NLRP3 activators disrupt the endocytic AP2 complex and plasma membrane signaling

doi: 10.64898/2026.02.24.707400

Figure Lengend Snippet: (A) Volcano-plot of streptavidin-bead-based pulldown from LPS-primed ASC -/- iBMDMs treated for 90 min with 100 µM biotin-Dynasore to 100 µM biotin-Dyngo-4a, comparing log2-transformed label-free quantification (LFQ)-intensities student’s t-test fold-enrichment and statistical significance; n = 3. (B) Heatmap showing log2-transformed LFQ-intensities of top-10 enriched interactors in cells treated with biotin-Dynasore compared to biotin-Dyngo-4a across compound concentrations (200, 100, 40, 10, 1, 0 µM biotin-Dynasore; 100, 50, 10, 1, 0 µM biotin-Dyngo-4a). (C) Fold IL-1β release of LPS-primed wt Balb/c iBMDMs 48 h after siRNA-mediated knockdown (5 nM) of the indicated genes stimulated with Nigericin (10 µM) or Dynasore (80 µM); n = 3. Statistics indicate significance by student’s two-tailed t-test (P < 0.01)). (D) Structure prediction of Dynasore in complex with hNME1 with Alphafold3. (E) IL-1β release of LPS-primed Balb/c iBMDMs expressing doxycycline-inducible NME1 fused to a mitochondrial targeting sequence peptide (COX8a). Statistics indicate significance by student’s two-tailed t-test (P < 0.01)). (F) IL-1β release of LPS-primed Balb/c iBMDMs expressing dominant negative Histidine118 mutants of NME2 in response to Dyngo-4a (80 µM) or Dynasore (80 µM). Statistics indicate significance by student’s two-tailed t-test; n = 3. (G-J) Phosphoproteomics analysis of inflammasome-stimulated Nlrp3 -/- iBMDMs. (G) Reference profiles for Pearson’s correlation clustering analysis of log2-transfomred, z-scored 8788 significantly regulated phosphosites across all conditions (One-way ANOVA, S 0 =1, FDR = 0.01) to determine top 500 downregulated phosphosites upon activation as shown in heatmap below. (H) As in (G) for upregulated phosphosites. (I) Enrichment analysis for Uniprot keywords using Fisher exact test on top 500 down- or (J) upregulated phosphosites compared to all 34530 identified phosphopeptides with an enrichment factor > 2.

Article Snippet: For spatial proteomics experiments, per replicate, approximately 100×10 6 Casp1/11 -/- or Asc -/- SILAC-heavy or -light labelled iBMDMs were primed with 1 μg/ml LPS-EB or LPS-EB Ultrapure from E. coli 0111:B4 (InvivoGen, #tlrl-eblps or #tlrl-3pelps) in CM for 4 h at 37°C, 5% CO 2 , followed by stimulation in serum free conditions with either 10 μM Nigericin (NI; Thermo Fisher Scientific, #N1495), 60 μg/ml R837/Imiquimod (InvivoGen, #tlrl-imqs-1), 10 μM monensin (Merck, #M5273), Mdivi-1 (Selleckchem, #S7162), K + -free Hank’s balanced salt solution (HBSS), 160 μM Dynasore (DS; Selleckchem, #S8047), 80 μM Dyngo-4a (DN; Selleckchem, #S7163), or serum-free DMEM as control.

Techniques: Transformation Assay, Quantitative Proteomics, Knockdown, Two Tailed Test, Expressing, Sequencing, Dominant Negative Mutation, Phospho-proteomics, Activation Assay

Journal: bioRxiv

Article Title: NLRP3 activators disrupt the endocytic AP2 complex and plasma membrane signaling

doi: 10.64898/2026.02.24.707400

Figure Lengend Snippet: (A) Fluorescent microscopy of SNAP-β 2 AR-HEK293 cells at baseline (0 min) and 20 minutes after stimulation with 1 µM Isoproterenol +/- 30 µM Dyngo-4a, 60 µM Dynasore or 60 µg/ml R837. (B) Maximum of compound-induced (1 µM Isoproterenol, 60 µM Dynasore, 30 µM Dyngo-4a or 60 µg/ml R837) internalization of overexpressed SNAP-β 2 AR in HEK293 cells relative to buffer between 40 and 50 min measured as diffusion-enhanced resonance energy transfer (DERET). (C) Representative Over-time DERET of isoproterenol (100 nM/1 µM) induced SNAP-β 2 AR internalization in HEK293 cells subsequent to 30 min stimulation with 60 µM Dynasore, 30 µM Dyngo-4a or 60 µg/ml R837(left) and quantification as maximal response relative to buffer between 40 and 50 minutes shown as the mean ± SEM of four independent experiments (right). Statistics indicate significance by student’s two-tailed t-test. (D) Representative Over-time (left) isoproterenol-induced recruitment of β-arrestin 2 to β 2 V 2 in HEK293 cells measured as BRET between Flag-β 2 V 2 -YFP and Flag-β 2 V 2 -YFP after pre-stimulation with 60 µM Dynasore, 30 µM Dyngo-4a or 60 µg/ml R837or vehicle and quantification as maximal BRET amplitude, shown as the mean ± SEM of three independent experiments (right). Statistics indicate significance by student’s two-tailed t-test. (E) Over-time (left) and maximal (right) CCL19-induced recruitment of β-arrestin 1 to CCR7 in HEK293 cells pre-stimulated 60 µM Dynasore, 10 µM Nigericin, 60 µg/ml R837 or vehicle controls, quantified via bioluminescence resonance energy transfer (BRET). (F) Relative over-time Förster resonance energy transfer (FRET) in HEK293T cells expressing the dual biosensor mlCNBD-FRET after addition of norepinephrine (NE) in the indicated concentrations subsequent to preincubation with either 1% DMSO, 60 µM Dynasore, 30 µM Dyngo-4a; peak cAMP production in response to norepinephrine treatment in indicated concentrations (NE). Cells were pre-treated with DMSO, Dynasore, Dyngo-4a for 30 min before the experiment. (G) Representative Over-time dynamic mass redistribution in HEK293 cells stably expressing CCR7, induced by stimulation with 1 µM CCL19 subsequent to 30 min stimulation with 60 µM Dynasore, 30 µM Dyngo-4a or 60 µg/ml R837 (left) and quantification as the maximum wavelength shift as mean ± SEM of at least three independent experiments. (H) Velocity in [µm/min] and directionality of 50 dendritic cells per replicate during in-vitro 3D collagen migration assay over a timespan of 3 h after treatment with 1% DMSO, 160 µM Dynasore, 10 µM Dyngo-4a or 10 µM MCC-950; n = 4. Statistics indicate significance by Kruskal-Wallis test (n.s. = not significant, **** = P < 0.0001).

Article Snippet: For spatial proteomics experiments, per replicate, approximately 100×10 6 Casp1/11 -/- or Asc -/- SILAC-heavy or -light labelled iBMDMs were primed with 1 μg/ml LPS-EB or LPS-EB Ultrapure from E. coli 0111:B4 (InvivoGen, #tlrl-eblps or #tlrl-3pelps) in CM for 4 h at 37°C, 5% CO 2 , followed by stimulation in serum free conditions with either 10 μM Nigericin (NI; Thermo Fisher Scientific, #N1495), 60 μg/ml R837/Imiquimod (InvivoGen, #tlrl-imqs-1), 10 μM monensin (Merck, #M5273), Mdivi-1 (Selleckchem, #S7162), K + -free Hank’s balanced salt solution (HBSS), 160 μM Dynasore (DS; Selleckchem, #S8047), 80 μM Dyngo-4a (DN; Selleckchem, #S7163), or serum-free DMEM as control.

Techniques: Microscopy, Diffusion-based Assay, Förster Resonance Energy Transfer, Two Tailed Test, Bioluminescence Resonance Energy Transfer, Expressing, Stable Transfection, In Vitro, Migration

Journal: bioRxiv

Article Title: NLRP3 activators disrupt the endocytic AP2 complex and plasma membrane signaling

doi: 10.64898/2026.02.24.707400

Figure Lengend Snippet: (A) Representative over-time DERET of SNAP- β 2 -AR internalization in HEK293 cells overexpressing SNAP- β 2 -AR subsequent to stimulation with 60 µM Dynasore, 30 µM Dyngo-4a, 60 µg/ml R837 or 1 µM Isoproterenol. Data are shown as the mean ± SD of one representative experiment mean ± SD of one representative experiment. (B) Representative over-time DERET of 100 nM isoproterenol induced SNAP- β 2 -AR internalization in HEK293 cells overexpressing SNAP- β 2 -AR subsequent to pre-stimulation with 60 µM Dynasore, 30 µM Dyngo-4a, 60 µg/ml R837 Data are shown as the mean. (C) Over-time cAMP production in response to norepinephrine treatment in indicated concentrations (NE). Cells were treated with DMSO or R837 (60 µg/ml) for 30 min before the experiment. (D) Maximal isoproterenol-induced recruitment of nLuc-tagged β-arrestin 2 to wt - β 2 -AR in HEK293 cells and individual over-time traces measured as BRET between nLuc- β arrestin 2 and a plasma-membrane mVenus-tagged kRas serving as BRET acceptor after pre-stimulation with 60 µM Dynasore, 30 µM Dyngo-4a or 60 µg/ml R837or vehicle of three independent experiments (right). (E) IL-1β release of dendritic cells stimulated with 80 µM Dynasore, 30 µg/ml R837, 10 µM Dyngo-4a or in combination with 10 µM MCC-950 for 90 min; n = 3. Statistics indicate significance by student’s two-tailed t-test (**** = P < 0.0001). (F) Immunofluorescence of ASC in BMDCs treated with 80 µM Dynasore, 10 µM MCC- 950, a combination thereof or vehicle control for 90 min. (G) Velocity in [µm/min] and directionality of dendritic cells per replicate during in-vitro 3D collagen migration assay over a timespan of 3 h after treatment with 1% DMSO, 30 µg/ml R837 or 30 µg/ml R837 + 10 µM MCC-950. Statistics indicate significance by Kruskal-Wallis test (n.s. = not significant, **** = P < 0.0001).

Article Snippet: For spatial proteomics experiments, per replicate, approximately 100×10 6 Casp1/11 -/- or Asc -/- SILAC-heavy or -light labelled iBMDMs were primed with 1 μg/ml LPS-EB or LPS-EB Ultrapure from E. coli 0111:B4 (InvivoGen, #tlrl-eblps or #tlrl-3pelps) in CM for 4 h at 37°C, 5% CO 2 , followed by stimulation in serum free conditions with either 10 μM Nigericin (NI; Thermo Fisher Scientific, #N1495), 60 μg/ml R837/Imiquimod (InvivoGen, #tlrl-imqs-1), 10 μM monensin (Merck, #M5273), Mdivi-1 (Selleckchem, #S7162), K + -free Hank’s balanced salt solution (HBSS), 160 μM Dynasore (DS; Selleckchem, #S8047), 80 μM Dyngo-4a (DN; Selleckchem, #S7163), or serum-free DMEM as control.

Techniques: Clinical Proteomics, Membrane, Two Tailed Test, Immunofluorescence, Control, In Vitro, Migration