Journal: medRxiv

Article Title: Airborne particulate matter enhances with monosodium urate crystals the secretion of IL-1β by human immune cells

doi: 10.64898/2026.02.26.26347218

Figure Lengend Snippet: Innate immune cell phenotype and IL-1 β expression after exposure of animals to CB+O 3 . Absolute counts for A) lavage neutrophils B) macrophages and C) particle-containing macrophages in lavage, n=4-6 per experiment. Evidence of NLRP3-inflammasome activation in the lungs after MSU injection and air pollution exposure day 8 post MSUc injection (D) pro-IL-1□ mRNA expression and E) IL-1□ concentration in lung lavage fluid, n=4-6 per experiment. * P <0.05, ** P <0.005, **** P <0.00005.

Article Snippet: NLRP3-inflammasome inhibitor MCC950 was purchased from Invivogen and used at 2 nmol/l.

Techniques: Expressing, Activation Assay, Injection, Concentration Assay

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Enhanced CDRs correlated with prostate cancer prognosis. (A) Visualization of core genes' dependency in different prostate cancer cells with CRISPR-Cas9 and siRNA screening. (B, C) Survival analysis of CDRs in different CRPC patient cohorts. (D) The relative expression of CDRs in normal prostate tissues and prostate cancer tissues in the TCGA-PRAD cohort. Gleason score and tumor stage correlation analysis of CDRs in different prostate cancer patient cohorts. All prostate patient cohorts were indicated in the corresponding panels. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2).

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: CRISPR, Expressing, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs linked with neuroendocrine features in prostate cancer cohorts. (A) The Venn diagram illustrates the genes specifically enhanced in prostate cancer compared with normal prostate tissues and NEPC compared with adenocarcinoma. (B, C) The relative expression of CDRs in NEPC compared with adenocarcinoma in different prostate cancer cohorts. (D) Correlation analysis of CDRs with NE score. The red indicates the higher NE score. (E, F) Pearson correlation analysis of CDRs with NE score CRPC cohort. (G) The heatmap indicates the relative expression of CDRs and AR, as well as KLK3, a transcription activity indication of AR. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). NEPC, neuroendocrine prostate cancer; NE, neuroendocrine; AR, androgen receptor; KLK3, kallikrein-related peptidase 3.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Expressing, Activity Assay, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs ablation suppressed prostate cancer cell proliferation. (A) The circuit diagram illustrates the correlation among CDRs co-expressed gene sets. (B) Venn analysis of the overlap of CDRs co-expressed genes. (C) The heatmap shows the relative expression of CDRs overlapped co-expressed genes in patients with NE signature high and low groups. (D – F) The heatmap illustrates the correlation of CDRs co-expressed genes with CDRs in ADPC (D) and CRPC (E, F) patient cohorts. (G, H) KEGG pathway analysis (G) and GSEA analysis (H) of enriched biological processes of CDRs and CDRs co-expressed genes. (I, J) The diagram illustrates the working model of CRISPR-Cas13 for RNA silencing and knockdown efficiency of CDRs with the corresponding gRNAs. (K, L) Cell growth assays indicate the impact of CDRs knockdown on the viability of different prostate cancer cells. (M) Western blotting analysis of the expression of cell cycle-regulated genes, including CCND1, CDK1, and p-CDK1, after CDRs knockdown. ∗∗ p < 0.01. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). NE, neuroendocrine; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis; CCND1, cyclin D1; CDK1, cyclin-dependent kinase 1.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Expressing, CRISPR, Knockdown, Western Blot, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs were transcriptionally regulated by the RB1/E2F1 axis in prostate cancer. (A) The diagrams show the canonical E2F1 motif location within the promoter of CDRs. (B) ChIP-sequencing E2F1 enrichment peak within the promoter of CDRs. (C) The relative enrichment of E2F1 within the promoter of CDRs was determined using standard ChIP-qPCR. (D) ChIP-sequencing peaks show the relative enrichment of E2F1 in CDRs' promoters after RB1 is known. (E – G) The relative expression of CDRs in CRPC patients with different RB1 deletion status. (H) CDRs expression in patients with RB1 deletion mutations from different prostate cancer cohorts. (I) qRT-PCR detected the relative expression of CDRs after RB1 or E2F1 knockdown with CRISPR-Cas13. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). RB1, retinoblastoma tumor suppressor 1; E2F1, early 2 factor 1; ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time PCR.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: ChIP-sequencing, ChIP-qPCR, Expressing, Quantitative RT-PCR, Knockdown, CRISPR, Ubiquitin Proteomics, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Virtual screening identified compounds that suppressed advanced prostate cancer. (A) The diagram illustrates structure-based virtual screening strategies for CDRs-targeted compounds. (B) The heatmap shows the binding affinity of compounds with CDRs. The red indicated a higher binding affinity of compounds with the corresponding compounds. (C, D) Cell viability assays were used to determine the tumor suppressive effect of compounds with high CDRs-binding affinity in different prostate cancer cell models. (E – G) The 2D structure of Q199, XDD60, and A79, which exhibits the most significant anti-tumor efficacy in prostate cancer cell models. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2).

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Binding Assay, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Compounds targeting CDRs exhibited superior anti-tumor efficacy compared with AR antagonists. (A – E) The tumor cell growth inhibition effects of different dosages of Q199, XDD60, and A79, as well as enzalutamide, were determined with CCK-8 assays (A–D), and the IC 50 of each agent was calculated with three independent experiments (E). (F, G) The histograms show the relative cell viability after being treated with 5 M of Q199, XDD60, or A79 alone, or a combination. (H) The Venn diagram shows the overlap of Q199, XDD60, and A79 potential targets predicted with SwissTargetPrediction ( http://swisstargetprediction.ch/ ). Molecular docking shows the binding of CDRs with Q199, XDD60, and A79. (I) The lowest binding (LB) affinity of CDRs with Q199, XDD60, and A79. ns, not significant. ∗∗ p < 0.01. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). AR, androgen receptor.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Inhibition, CCK-8 Assay, Binding Assay, Ubiquitin Proteomics

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

Journal: bioRxiv

Article Title: Digest before Ingest: Early Recruitment of Membrane-bound DNaseX to Phagocytic Cups in Macrophages

doi: 10.64898/2026.02.23.707500

Figure Lengend Snippet: (A) Surface-immobilized nuclease sensor (SNS) reports DNase activity in the PCs of macrophages. Microbeads are immobilized on a glass substrate to elicit phagocytosis. The entire surface, including the microbeads, is coated with SNS, a double-stranded DNA (dsDNA) labeled with a quencher-dye pair. SNS becomes fluorescent upon degradation by DNase. (B) Human THP-1 macrophages were plated on the DNase-reporting platform, where fluorescence signals appeared in ring patterns, co-localizing with F-actin surrounding the microbeads. (C) Comparison of SNS fluorescence intensities on microbeads underneath cell bodies versus those outside of cells. (**** P <0.0001; Each data point represents one PC; n = 3 experiments; Error bars indicate SD). (D) Confocal 3D scanning revealed that the SNS signal was localized on the surface of microbeads. ( E-F ) dsDNA-dye coated on microbeads exhibited a decrease in fluorescence intensities beneath macrophages. ( G-H ) ssDNA-dye coated on microbeads exhibited a decrease in fluorescence intensities beneath macrophages. (I) Time-lapse co-imaging of F-actin and SNS signals in the PCs of live macrophages. (J) Signal intensity curves of F-actin and SNS on one microbead. The time gap Δt between the starting times of these two signals is defined as the emergence time of DNase activity in the PCs. (K) Emergence time of DNase activity in the PCs was statistically estimated to be 48±33 sec after the PC formation indicated by F-actin signal.

Article Snippet: Human monocytes were treated with 100 ng/ml macrophage colony stimulating factor (M-CSF; 216-MCC; R&D Systems) for 48 hours.

Techniques: Activity Assay, Labeling, Fluorescence, Comparison, Imaging

Journal: bioRxiv

Article Title: Digest before Ingest: Early Recruitment of Membrane-bound DNaseX to Phagocytic Cups in Macrophages

doi: 10.64898/2026.02.23.707500

Figure Lengend Snippet: ( A-C ) DNase activities were consistently observed in the PCs of mouse RAW macrophages, human THP-1 macrophages and human monocyte-derived macrophages. The PCs, marked by F-actin structures, were formed on microbeads of various sizes (0.3, 1.1 and 3.0 µm). ( D ) DNase activity was consistently observed in the PCs of M0, M1, and M2 THP-1 macrophage subtypes. ( E-G ) SNS signal intensities in the PCs of the three types of macrophages. (Each data point represents one PC; n = 3 experiments; Error bars indicate SD). ( H ) SNS signal intensities in the PCs of the three subtypes of THP-1 macrophages. (Each data point represents one PC; n = 3 experiments; Error bars indicate SD)

Article Snippet: Human monocytes were treated with 100 ng/ml macrophage colony stimulating factor (M-CSF; 216-MCC; R&D Systems) for 48 hours.

Techniques: Derivative Assay, Activity Assay

Journal: bioRxiv

Article Title: Digest before Ingest: Early Recruitment of Membrane-bound DNaseX to Phagocytic Cups in Macrophages

doi: 10.64898/2026.02.23.707500

Figure Lengend Snippet: (A) F-actin and SNS signals in the PCs of THP-1 macrophages treated with PI-PLC, which cleaves GPI linkers of the putative membrane-bound DNase in the PCs. (B) PI-PLC treatment significantly reduced SNS signals in the PCs, indicating marked decreases of DNase activities. (**** P <0.0001; Each data point represents one PC; n = 3 experiments; Error bars indicate SD). (C) F-actin and SNS signals in the PCs of THP-1 macrophages with DNaseX knocked down by siRNA interference. (D) DNaseX knockdown significantly reduced SNS signals in the PCs (**** P <0.0001; Each data point represents one PC; n = 3 experiments; Error bars indicate SD). (E) Co-imaging of immunostained DNaseX, F-actin and SNS signals in the PCs of THP-1 macrophages.

Article Snippet: Human monocytes were treated with 100 ng/ml macrophage colony stimulating factor (M-CSF; 216-MCC; R&D Systems) for 48 hours.

Techniques: Membrane, Knockdown, Imaging

Journal: bioRxiv

Article Title: Digest before Ingest: Early Recruitment of Membrane-bound DNaseX to Phagocytic Cups in Macrophages

doi: 10.64898/2026.02.23.707500

Figure Lengend Snippet: ( A-B ) SNS signal, F-actin and immunostained DNaseX on the ventral surfaces of adherent THP-1 macrophages, which were detached from a culture flask by either EDTA (A) or trypsin (B) prior to cell plating. The cells were incubated on the SNS surfaces for 1 h. ( C-D ) DNase activities in the PCs indicated by SNS signals with 20 min, 60 min and 90 min incubation times, respectively. The macrophages were detached by either EDTA (C) or trypsin (D). ( E ) SNS signal intensities in PCs. (* P <0.05; ns P>0.05; Each data point represents one PC; n = 3 experiments; Error bars indicate SD)

Article Snippet: Human monocytes were treated with 100 ng/ml macrophage colony stimulating factor (M-CSF; 216-MCC; R&D Systems) for 48 hours.

Techniques: Incubation

Journal: bioRxiv

Article Title: Digest before Ingest: Early Recruitment of Membrane-bound DNaseX to Phagocytic Cups in Macrophages

doi: 10.64898/2026.02.23.707500

Figure Lengend Snippet: ( A ) Images of F-actin, DNaseX and SNS signals in the PCs with the macrophages treated with DMSO (control), 100 µM CK666 or 1 µM Cytochalasin D, respectively. ( B-D ) Signal intensities of F-actin, SNS and DNaseX signals in the PCs with the above treatments. (**** P <0.0001; ** P <0.01; Each data point represents one PC; n = 3 experiments; Error bars indicate SD).

Article Snippet: Human monocytes were treated with 100 ng/ml macrophage colony stimulating factor (M-CSF; 216-MCC; R&D Systems) for 48 hours.

Techniques: Control

Journal: bioRxiv

Article Title: Digest before Ingest: Early Recruitment of Membrane-bound DNaseX to Phagocytic Cups in Macrophages

doi: 10.64898/2026.02.23.707500

Figure Lengend Snippet: (A) eDNA structures in S. Aureus biofilms. The eDNA was stained with DITO-1. (B) RAW macrophages incubated on S. Aureus biofilms for 30 min. The filamentous eDNA structures were degraded under cell bodies. (C) Line-profile analysis of eDNA degradation regions indicated by blue lines in (B), which are shown to have sharp boundaries with ∼1 µm transition from undegraded region to degraded region, suggesting that the eDNA degradation was mediated by non-diffusive DNase. (D) Time-series images of eDNA degradation by a RAW macrophage. Related to Video 3. The eDNA filament (indicated by a blue arrow) became gradually shortened during the degradation. (F) Analysis of eDNA degradation by macrophages. The degradation process typically spans 20-30 minutes.

Article Snippet: Human monocytes were treated with 100 ng/ml macrophage colony stimulating factor (M-CSF; 216-MCC; R&D Systems) for 48 hours.

Techniques: Staining, Incubation

Journal: bioRxiv

Article Title: Digest before Ingest: Early Recruitment of Membrane-bound DNaseX to Phagocytic Cups in Macrophages

doi: 10.64898/2026.02.23.707500

Figure Lengend Snippet:

Article Snippet: Human monocytes were treated with 100 ng/ml macrophage colony stimulating factor (M-CSF; 216-MCC; R&D Systems) for 48 hours.

Techniques: