Fig 2: Role of platelet GM-CSF and C3 in neutrophil-DNA release. a–c Isolated human platelets and neutrophils were incubated together or by themselves for 30 min (at constant rotation and 37 °C) in the presence of TLR agonists [TLR7 (Loxo)—1 mM; TLR2 (Pam3CSK4, PAM)—10 μg/μL] or thrombin (IIa)—0.05 U/mL. GM-CSF release from a platelets (p = 0.8903, F = 0.2071, df = 3), b neutrophils (p = 0.7255, F = 0.4422, df = 3), and c platelets and neutrophils incubated together was measured by ELISA (p = 0.0200, F = 4.116, df = 3). The graphs represent the average fold change for each individual of n = 6 (3F; 3M) ± SD. d Confocal images of isolated human neutrophils treated with C3 (30 ng/mL) and neutrophils treated with C3 in the presence of GM-CSF (25 ng/mL). Neutrophils were treated in HEPES-modified Tyrode’s buffer (0.04 × 105 neutrophils/μL) for 30 min at 37 °C, and constant rotation (aggregometer). At the end, cells were fixed and stained [CD41-FITC-platelets (green); H4-AF637-histone (red); DAPI-DNA (blue)]. Neutrophil-DNA aggregates were visualized by confocal microscopy. Images are representatives of n = 4 different donors. C3-treated neutrophils from healthy donors release their DNA and form large aggregates. e Quantitation of (d). The graph (n = 4, 2F, 2M) is represented as average (p < 0.0001, F = 71.42, df = 3) ± SD. Significance in (a–c, e) was assessed using ANOVA followed by Bonferroni multiple comparison test and star symbol (*) indicates p < 0.05. Source data are provided as a Source Data file

Fig 3: Silencing of HDAC4 suppresses IL‐13‐evoked inflammation in hNECs. (A) RT‐qPCR and western blot analysis of the interference efficacy of HDAC4‐targeted siRNAs. (B) Levels of histamine and IgE were detected by ELISA assay. (C) ELISA assay examined GM‐CSF, eotaxin, IL‐1β and IL‐6 levels. HDAC4, histone deacetylase 4; IL‐13, interleukin‐13; IgE, Immunoglobulin E; IL‐1β, interleukin‐1 beta; IL‐6, interleukin 6; GM‐CSF, granulocyte‐macrophage colony‐stimulating factor; RT‐qPCR, quantitative reverse‐transcription polymerase chain reaction

Fig 4: RUNX2 is activated by matrix stiffness via mechanotransduction(A) Immunoblot of RUNX2 in patient-derived xenograft (PDX) primary cells and breast cancer cell lines, preconditioned on soft and stiff hydrogels for 7 days (representative of n = 2 biological replicates).(B) Immunoblot of RUNX2 in SUM159 and T47D cells cultured for 7 days in 3D matrix consisting of soft 1.0 mg/mL rat-tail collagen-I, or stiff 1.0 mg/mL rat-tail collagen-I crosslinked with PEG-di(NHS) to stiffen the collagen lattice without changing ligand density (representative of n = 3 biological replicates).(C) qRT-PCR of 4 RUNX2 target genes in SUM159 cells preconditioned for 7 days on soft and stiff hydrogels with non-targeting small hairpin RNA (shRNA) (GIPZ), or on stiff hydrogels with 2 shRNAs targeting RUNX2 (n = 3 biological replicates). Data are means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001; 1-way ANOVA with Tukey’s multiple comparisons test.(D) qRT-PCR of RUNX2 in SUM159 cells preconditioned for 7 days on soft and stiff hydrogels with non-targeting shRNA (GIPZ), or on stiff hydrogels with 2 shRNAs targeting RUNX2 (n = 3 biological replicates). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; 1-way ANOVA with Sidak’s multiple comparisons test.(E) qRT-PCR of RUNX2 and 4 target genes, plus CTGF (YAP target) and PLIN1 (adipogenic biomarker) in SUM159 cells preconditioned, as indicated (n = 3 biological replicates). Data are means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001; 1-way ANOVA with Tukey’s multiple comparisons test.(F) qRT-PCR of the RUNX2 gene target OPN, and 3 YAP targets—CTGF, CYR61, and ANKRD1—in SUM159 cells preconditioned as indicated, and without media change for 48 h before sample collection (n = 3 biological replicates). Data are means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001; 1-way ANOVA with Tukey’s multiple comparisons test.(G) Immunoblot of RUNX2, ERK, and pERK in SUM159 cells stably expressing lentiviral shRUNX2 or GIPZ (non-targeting control), preconditioned for 7 days on soft or stiff hydrogels (representative of n = 3 biological replicates).(H) Immunoblot of pERK and ERK in SUM159 cells cultured on stiff hydrogels with 20 µM PD98059, 30 µM blebbistatin, 100 nM dasatinib, 1 µM Faki14, or DMSO for 1 h before lysis (representative of n = 3 biological replicates).(I) Immunofluorescence of pFAK, paxillin, and F-actin in SUM159 cells cultured on stiff or soft hydrogels for 7 days. Scale bar, 10 µm.(J) Immunoblot of OPN in SUM159 cells preconditioned for 7 days on soft and stiff hydrogels with non-targeting shRNA (GIPZ), on stiff hydrogels with 2 shRNAs targeting RUNX2, on soft hydrogels with constitutively active MEK-DD expression, or on stiff hydrogels with MEK inhibitor PD98059 (20 µM) (representative of n = 3 biological replicates).(K and L) qRT-PCR of OPN (K) and GM-CSF (L) in SUM159 cells preconditioned for 7 days on stiff hydrogels conjugated with either poly D-lysine (PDL) to reduce integrin binding, or collagen conjugated with DMSO (control), 30 µM blebbistatin, 100 nM dasatinib, or 1 µM Faki14 in media changed every other day (n = 3 biological replicates). Data are means ± SEMs and normalized to 7-day stiff controls. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; 1-way ANOVA with Sidak’s multiple comparisons test.(M) Immunoblot showing phospho-AKT levels in SUM159 cells treated with DMSO (control) or AKT inhibitor (1 µM MK-2206) (representative of n = 2 biological replicates).(N) qRT-PCR of RUNX2 target genes in SUM159 cells preconditioned for 7 days on stiff hydrogels and treated with DMSO or 1 µM AKT inhibitor MK-2206 (n = 3 biological replicates). Data are means ± SEMs. Multiple t test with Holm-Sidak multiple comparisons; adjusted p values are not significant (n.s.).

Fig 5: Effect of TQ and TQ-loaded LNCs on immune system response. Levels of (A) GM-CSF (ng mL−1), and (B) TFN-α (pg mL−1) for the different treated groups (a: P < 0.05 versus control group, b: P < 0.05 versus TQ group).