Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: Intercellular communication analysis predicts IL-1β as a candidate upstream signal potentially associated with neutrophils. ( A ) Chord diagram showing predicted ligand–target interactions between sender cell populations and receiver SSPCs in the NonU microenvironment. Immune cell populations contributed prominently to the inferred SSPC-directed signaling network. ( B ) Dot plot showing the top predicted ligands received by SSPCs and their expression patterns across potential sender cell populations. Dot size indicates the percentage of ligand-expressing cells, and color indicates average expression. ( C ) Heatmap showing log-fold changes in prioritized ligands in their corresponding sender populations when comparing NonU with Frac conditions. ( D ) Heatmap showing the predicted regulatory potential of prioritized ligands on SSPC target genes. Candidate transcription factors are indicated. ( E ) GO enrichment analysis of predicted IL-1β target genes in SSPCs. Ferroptosis-related terms are highlighted. ( F ) KEGG enrichment analysis of predicted IL-1β target genes in SSPCs. Ferroptosis and fatty acid metabolism pathways are highlighted. Red arrows and red-colored labels indicate representative ligands, cell populations, target genes, transcription factors, or enriched terms highlighted and discussed in the main text.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: IL-1β induces ferroptotic stress and impairs SSPC function in vitro. ( A ) Schematic overview of SSPC isolation by fluorescence-activated cell sorting and subsequent in vitro validation experiments. ( B ) Flow cytometry gating strategy for isolating Lin − /DPP4 + SSPCs from mouse callus tissue at 7 days post-fracture. ( C ) Representative Western blot images showing the expression of ACSL4, COX2, 4-HNE, NF-κB p65, and EPAS1 in SSPCs treated with vehicle, erastin, or IL-1β. β-actin was used as the loading control. ( D ) Quantification of Western blot protein expression levels shown in panel C ( n = 3). ( E ) Representative BODIPY 581/591 C11 fluorescence images showing lipid peroxidation in SSPCs. Oxidized lipids are indicated by increased green fluorescence and reduced red fluorescence. Scale bar = 50 μm. ( F ) Representative FerroOrange fluorescence images showing intracellular Fe 2+ levels in SSPCs. Scale bar = 50 μm. ( G ) Quantification of the red/green fluorescence intensity ratio from BODIPY 581/591 C11 staining and the relative FerroOrange fluorescence intensity ( n = 3). A lower red/green ratio indicates increased lipid peroxidation. ( H ) Quantification of colony-forming unit efficiency and cell viability measured by CCK-8 assay ( n = 3). ( I ) qRT-PCR analysis of osteogenic, chondrogenic, and adipogenic marker gene expression ( n = 3). ( J ) Representative images of tri-lineage differentiation assays, including Alizarin Red S staining for osteogenesis, Alcian Blue staining for chondrogenesis, and Oil Red O staining for adipogenesis. Scale bar = 200 μm. ( K ) qRT-PCR analysis of osteogenic, chondrogenic, and adipogenic marker gene expression, including Runx2, Opn, Acan, Col2a1, Pparg, and Cebpa ( n = 3). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: In Vitro, Isolation, Fluorescence, FACS, Biomarker Discovery, Flow Cytometry, Western Blot, Expressing, Control, Staining, CCK-8 Assay, Quantitative RT-PCR, Marker, Gene Expression, Comparison

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: Pharmacological EPAS1 inhibition attenuates IL-1β-induced ferroptotic stress and differentiation impairment in primary SSPCs. ( A ) Representative images of Alcian Blue and Alizarin Red S staining in primary SSPCs treated with vehicle control, IL-1β, IL-1β plus PT2385, or PT2385 alone. Scale bars = 200 μm. ( B ) Representative Western blot images showing ACSL4, 4-HNE, EPAS1, and NF-κB p65 protein levels in SSPCs under the indicated treatments. GAPDH was used as the loading control. ( C ) qRT-PCR analysis of differentiation-related marker genes, including the chondrogenic markers Sox9 and Acan and the osteogenic markers Runx2 and Opn (n = 3). ( D ) Quantification of Western blot protein expression levels shown in panel ( B ) (n = 3). ( E ) Representative BODIPY 581/591 C11 fluorescence images showing lipid peroxidation in SSPCs. Red fluorescence indicates non-oxidized lipid signal, and green fluorescence indicates oxidized lipid signal. Scale bars = 50 μm. ( F ) Representative FerroOrange fluorescence images showing intracellular Fe 2+ levels in SSPCs under the indicated treatments. Scale bars = 50 μm. ( G ) Quantification of the red/green fluorescence intensity ratio from BODIPY 581/591 C11 staining (n = 3). A lower red/green ratio indicates increased lipid peroxidation. ( H ) Quantification of relative FerroOrange fluorescence intensity (n = 3). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Inhibition, Staining, Control, Western Blot, Quantitative RT-PCR, Marker, Expressing, Fluorescence, Comparison

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: EPAS1 inhibition attenuates IL-1β-impaired bone regeneration in vivo. ( A ) Representative micro-CT three-dimensional reconstructions and micro-CT sectional images of fractured mouse femurs from vehicle control, IL-1β, IL-1β plus PT2385, and PT2385 alone groups at 28 days post-fracture. Scale bars = 1 mm. ( B ) Quantitative micro-CT analysis of bone volume (BV) and bone volume fraction (BV/TV) in the fracture callus region. ( C ) Quantitative histomorphometric analysis of bone area and callus area among the four treatment groups. ( D ) Representative Safranin O/Fast Green staining images of the fracture callus region showing callus organization, cartilage matrix, and newly formed bone tissue under the indicated treatments. Scale bars = 500 μm. Data are presented as mean ± SD; n = 6 mice per group. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Inhibition, In Vivo, Micro-CT, Control, Staining, Comparison

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: Mendelian randomization provides exploratory genetic support linking IL-1β and EPAS1 to human bone nonunion risk. ( A ) Forest plot summarizing inverse-variance weighted Mendelian randomization estimates for the tested exposures and bone nonunion risk. Odds ratios and 95% confidence intervals are shown. ( B ) Scatter plots showing the associations between SNP effects on EPAS1 expression or IL-1β levels and SNP effects on bone nonunion risk. Lines indicate estimates from different Mendelian randomization methods. ( C ) Single-SNP forest plots showing individual SNP estimates for EPAS1 and IL-1β. The combined inverse-variance weighted estimates are shown at the bottom. ( D ) Leave-one-out analyses for EPAS1 and IL-1β, performed by sequentially excluding one SNP at a time. ( E ) Funnel plots used to assess potential directional pleiotropy in the Mendelian randomization analyses for EPAS1 and IL-1β. Red colors, red lines, and numbers indicate representative annotations used to highlight key features discussed in the main text.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Expressing