Fig 1: SIRT1 inhibitor EX527 reverses the impacts of HDAC4 depletion on IL-13-primed hNECs. (A) Western blot analyzed the protein levels of SIRT1, p-NF-?B, and NF-?B. (B) Levels of histamine and IgE were detected by ELISA assay. (C) ELISA assay examined GM-CSF, eotaxin, IL-1ß, and IL-6 levels. (D) RT-qPCR and western blot analysis of MUC5AC expression. (E) MUC5AC expression was further tested by IF assay. GM-CSF, granulocyte-macrophage colony-stimulating factor; HDAC4, histone deacetylase 4; IL-13, interleukin-13; IgE, Immunoglobulin E; IL-1ß, interleukin-1 beta; IL-6, interleukin 6; MUC5AC, Mucin-5AC; p-NF-?B, phosphorylated nuclear factor-kappaB; NF-?B, nuclear factor-kappaB; RT-qPCR, quantitative reverse-transcription polymerase chain reaction
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-1ß and IL-6 levels. HDAC4, histone deacetylase 4; IL-13, interleukin-13; IgE, Immunoglobulin E; IL-1ß, 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.).
Supplier Page from Abcam for Human GM-CSF ELISA Kit (CSF2)