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 experiments in primary BMDMs, BMDCs or iBMDMs which required inhibition of pyroptosis, we used 10 μM MCC-950 (InvivoGen, #inh-mcc), 10 μM VX-765 (InvivoGen, #inh-vx765i-1) or serum-free DMEM as control unless stated otherwise.

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 experiments in primary BMDMs, BMDCs or iBMDMs which required inhibition of pyroptosis, we used 10 μM MCC-950 (InvivoGen, #inh-mcc), 10 μM VX-765 (InvivoGen, #inh-vx765i-1) or serum-free DMEM as control unless stated otherwise.

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 experiments in primary BMDMs, BMDCs or iBMDMs which required inhibition of pyroptosis, we used 10 μM MCC-950 (InvivoGen, #inh-mcc), 10 μM VX-765 (InvivoGen, #inh-vx765i-1) or serum-free DMEM as control unless stated otherwise.

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) 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 experiments in primary BMDMs, BMDCs or iBMDMs which required inhibition of pyroptosis, we used 10 μM MCC-950 (InvivoGen, #inh-mcc), 10 μM VX-765 (InvivoGen, #inh-vx765i-1) or serum-free DMEM as control unless stated otherwise.

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 experiments in primary BMDMs, BMDCs or iBMDMs which required inhibition of pyroptosis, we used 10 μM MCC-950 (InvivoGen, #inh-mcc), 10 μM VX-765 (InvivoGen, #inh-vx765i-1) or serum-free DMEM as control unless stated otherwise.

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