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) 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: Local albumin excess exacerbates Candida albicans-induced inflammasome activation linked with hyperinflammation during vulvovaginal candidiasis

doi: 10.64898/2026.01.22.700771

Figure Lengend Snippet: (A) Overview of NLRP3 inflammasome priming and activation. DAMPs = damage-associated molecular patterns. TLR = Toll-like receptor. PAMPs = pathogen-associated molecular pattern. (B) IL-1β and IL-18 release by hMDMs ( n = 14 donors). (C) IL-1β release by mBMDMs ( n = 10). (D) IL-1β release by hMDMs in the presence of the inflammasome inhibitors KCl ( n = 8 donors) or VX-765 ( n = 9 donors). (E) Percentage ASC-positive mBMDMs ( n = 3 uninfected , n = 4 infected). (F, G) IL-1β release of mBMDMs deficient in the NLRP3 receptor ( Nlrp3 −/− ), the adapter proteins ASC ( Pycard −/− ) or the proteolytic enzyme caspase 1 ( Casp1 −/− / Casp11 −/− ) ( n = 4) or deficient in gasdermin D ( Gsdmd −/− ) (G). Bars represent the mean + SEM with dots as individual biological replicates: Individual donors (B, D) or mice (C, E - G) from at least three independently conducted experiments. Statistical significance was determined using a two-way ANOVA including with Holm-Šídák post-hoc test. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001

Article Snippet: For inhibitor experiments, Anakinra (recombinant human IL-1Ra, 10 μg/mL, Kineret), potassium chloride (25 mM; Merck) or the caspase-1 inhibitor VX-765 (50 μg/mL; Invivogen) were added 1 h prior to infection.

Techniques: Activation Assay, Infection

Journal: Frontiers in Pharmacology

Article Title: Hyperoside ameliorates lupus nephritis by suppressing AKT1-mediated PANoptosis in podocytes: integrating network pharmacology and experimental validation

doi: 10.3389/fphar.2025.1726254

Figure Lengend Snippet: PANoptosis contributes to renal pathology in lupus nephritis. (A) Comparison of 24-h urinary protein levels between MRL/MpJ and MRL/lpr mice (n = 6). (B) Representative renal histology evaluated by H&E staining (n = 6). (C) TUNEL staining showing cell death in glomeruli (n = 6). (D) IHC analysis of podocyte markers (Podocalyxin, Nephrin, Synaptopodin, Podocin) in renal tissues. (E–G) Immunofluorescence staining of PANoptosis-related markers (NLRP3, RIPK3, Caspase-9) in kidney sections. (H) WB analysis of PANoptosis executor proteins (caspase-3, caspase-1, p-MLKL) in renal tissues, with bar graphs showing quantitative results relative to GAPDH (n = 3). (I) IF triple-staining of caspase-3, caspase-1, and p-MLKL in mouse kidney tissues. (J) Viability of MPC-5 cells treated with ICs at different concentrations and time points, measured by CCK-8 assay. (K) Flow cytometric analysis of cell death in MPC5 cells pretreated with VX-765 (10 μM), Nec-1 (20 μM), or Z-VAD (5 μM), followed by IC stimulation for 24 h. Bar graph shows the percentage of dead cells (n = 3). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, ns: not significant.

Article Snippet: The inhibitors VX-765 (HY-13205), Necrostatin-1 (Nec-1, HY-15760), and Z-VAD-FMK (Z-VAD, HY-15760) were sourced from MedChemExpress (MCE, USA).

Techniques: Comparison, Staining, TUNEL Assay, Immunofluorescence, CCK-8 Assay