Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A) Schematic of the primary mouse astrocyte cultures. (B) Representative immunofluorescence images of cultured astrocytes stained for GFAP, showing characteristic astrocyte morphology. (C) Overview of RNA-seq and capped small (cs)RNA-seq data generation from astrocytes. The schematic shows the typical distribution of csRNA-seq and RNA-seq at various genomic locations, which allows the identification of Transcriptional Start Site (TSSs) using HOMER2. (D) Volcano plot of RNA-seq differential expression in astrocytes treated with vehicle (Veh) or IL-1B (10 ng/mL, 1 h). (E) Pathway enrichment analysis of IL-1B-induced differentially expressed genes. (F) RT-qPCR validation of selected IL-1B-responsive genes in astrocytes

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: Immunofluorescence, Cell Culture, Staining, RNA Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Biomarker Discovery

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: Genome browser views showing RNA-seq signal at the Cxcl1, Ccl20, Csf2 , and Il6 loci in astrocytes treated with vehicle or IL-1B. These representative examples illustrate the robust transcriptional induction of canonical inflammatory genes following IL-1B stimulation. Gene models and genomic coordinates are shown in mm10 .

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A) Pairwise correlation analysis of csRNA-seq replicates from untreated and IL-1B-treated astrocytes. (B) Distribution of strand-specific csRNA-seq reads around GENCODE-annotated TSSs, showing strong enrichment at annotated transcriptional start sites. (C) Genomic annotation of astrocytes TSRs, partitioned across promoter, intronic, intergenic, and other genomic features. (D) Average chromatin profiles at promoter-distal TSRs, showing ATAC-seq and H3K27ac enrichment around transcribed regulatory elements. (E) Average csRNA-seq signal centered on promoter-distal ATAC-seq peaks, comparing transcribed and non-transcribed accessible regions (No Tx: 225,150 peaks, Tx: 14,836 peaks). (F) Average mCH profiles at promoter-proximal TSRs, promoter-distal transcribed TSRs, and non-transcribed distal accessible regions in astrocytes (data from ). (G) Distribution of NFIA and TEAD4 ChIP-seq peaks found overlapping TSRs and ATAC-seq peaks in astrocytes. (H)ChIP-seq read density for NFIA and TEAD4 centered on csRNA-seq defined TSRs, showing transcription factor binding immediately upstream of the primary TSS.

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A)Volcano plot of differentially regulated csRNA-seq signal at astrocyte TSRs following IL-1B stimulation, identifying induced and repressed regulatory elements. (B) Genome browser tracks depicting induction of promoter and distal regulatory enhancer transcription by csRNA-seq at the Ccl2 locus. (C) GREAT functional enrichment analysis of IL-1B induced TSRs. (D) Spatial clustering of IL-1B induced TSRs, depicting the density of TSRs in each category adjacent to Induced TSRs. (E) De novo motif enrichment analysis of IL-1B induced TSRs by HOMER. (F) Average csRNA-seq signal centered on promoter-distal NF-κB/p65 peaks, showing increased bidirectional transcription at p65-bound distal elements after IL-1B stimulation, consistent with eRNA induction. (G) Spatial density of NF-κB, AP1, and IRF motifs relative to TSRs (TF binding sites per bp per TSS), showing upstream enrichment of these motifs at IL-1B-induced TSRs compared with unchanged or repressed TSRs.

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: Functional Assay, Binding Assay

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A) Genome browser example of an IL-1B-induced locus (Cxcl10) in astrocytes, showing increased transcription initiation after stimulation. (B) Scatter plot comparing IL-1B-induced Log2 csRNA-seq changes at the promoter vs. RNA-seq changes across genes, highlighting genes regulated primarily at initiation (along x-axis) versus those showing stronger changes at the mRNA level (along y-axis). (C) Genome browser tracks at the Junb locus showing increased gene expression and RNAPII elongation in the gene body with limited change in promoter initiation and RNAPII promoter levels, consistent with regulation being mediated primarily at the level of transcription elongation rather than increased initiation. (D) RNAPII ChIP-seq levels at the promoters of IL-1B induced genes stratified by genes with minimal versus strong increases in csRNA-seq initiation activity. (E) Scatter plot comparing changes in overall TSR levels (Log2 Fold change, NT vs. IL-1B) versus their WIP score significance (the −Log10 p-value), identifying TSRs with altered initiation patterns independent of changes in total transcriptional output. (F) Representative examples of TSRs exhibiting a strong change in initiation pattern (top, WIP score −1.61, Lg10 p-value = 9.54e-05) versus a strong change in overall initiation levels with minimal change in initiation shape(WIP score −0.13, Log10 p-value = 0.84). (G) Scatter plot of TF motif enrichment in TSRs with significant changes in overall activity versus changes in TSS positions, highlighting differential associations of NF-κB, TEAD, and NFI motifs with these TSRs classes. (H) Venn diagram showing the overlap of ChIP-seq peaks for NFIA and TEAD4 before and after IL-1B stimulation in astrocytes. (I) Motif enrichment analysis of condition-specific (either Veh or IL-1B) TEAD4- and NFIA-bound regions, showing IL-1B-specific enrichment for inflammatory TF motifs, including NF-κB, IRF (IRF8), and AP1 (i.e. Fra1).

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: RNA Sequencing, Gene Expression, ChIP-sequencing, Activity Assay

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A) Venn diagram showing overlap between IL-1B-induced genes in astrocytes and KLA-induced genes in bone marrow-derived macrophages (BMDMs). (B) Venn diagram showing overlap between induced TSRs in astrocytes and BMDMs, revealing largely distinct stimulus-responsive enhancer landscapes despite partial overlap in induced genes. (C) Genome browser view of Tnfaip3 locus illustrating that astrocytes and BMDMs induce the same gene but use different enhancers upstream a shared promoter. (D) Top: Venn diagram showing overlap of NF-κB binding sites between activated astrocytes and BMDMs. Bottom: Fraction of astrocyte-specific, shared, and BMDM-specific p65 peaks associated with TSRs induced in astrocytes only, in both cell types, and in BMDM only. (E) Heatmap of de novo motif enrichment at cell type-specific induced TSRs, showing enrichment of lineage-associated motifs for each cell type. (F) Fraction of induced TSR classes bound by lineage-associated TFs, showing preferential association of astrocyte-induced TSRs with NFI and TEAD4 and macrophage-induced TSRs with PU.1 and CEBPα.

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: Derivative Assay, Binding Assay

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A) Average eRNA signal centered on astrocyte-specific, shared, and BMDM-specific NF-κBp65 peaks, showing cell type-matched induction of regulatory transcription at p65 bound sites. (B) Fraction of astrocyte-specific, shared, and BMDM-specific p65 peaks overlapping accessible chromatin regions in astrocytes or BMDMs, indicating that NF-κB recruitment occurs preferentially at cell-type-specific open chromatin regions. (C) Violin plots showing increase in ChIP-seq signal for p65, NFIA, TEAD4 at astrocyte IL-1B-induced TSRs and for p65, PU.1, and CEBPβ in macrophage KLA-induced TSRs.

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: ChIP-sequencing

Journal: Bioactive Materials

Article Title: Precisely regulated physically-crosslinked carriers enable synergetic release of bioactive factors for MSC-mediated cartilage regeneration

doi: 10.1016/j.bioactmat.2026.01.009

Figure Lengend Snippet: In vivo chondrogenesis of hydrogels incorporated with different combinations of PSF and KSF. (a) Alcian blue and Safranin O staining of different combinations after 21-day implantation. G1: 5 % β-sheet PSF +5 % β-sheet KSF; G2: 15 % β-sheet PSF +30 % β-sheet KSF; G3: 30 % β-sheet PSF +40 % β-sheet KSF; G4: 40 % β-sheet PSF +50 % β-sheet KSF. Scale bar = 200 μm. (b–d) Quantitative analysis of IL-1β, IL-6 and TNF-α surrounding defect cartilage 1 week and 3 weeks after operation. (e, f) Macroscopic and MRI observations of rat femoral condyles at week 6 and 12. Red circles and red arrows show the original defect zone under macroscope and MRI respectively. Scale bar = 1 mm. (g) Relative ratio of average optical density (versus G1) that shows the staining intensity of Alcian blue and Safranin O staining. (h) ICRS scoring of macroscopic evaluations. (i) The repaired cartilage using a nanoindentation instrument. Scale bar = 1 cm. (j, k) Reduced modulus and hardness of the regenerated cartilage. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: The suspension was centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was collected, and Elisa assay was performed following the manufacturer's instructions for rat IL-1β, IL-6, and TNF-α ELISA kits (Elabscience, China).

Techniques: In Vivo, Staining

Journal: Genes & Diseases

Article Title: Targeting MDK alleviates bone loss via dual regulation of osteogenic differentiation and inflammatory cytokine expression

doi: 10.1016/j.gendis.2025.101931

Figure Lengend Snippet: iMDK alleviates bone loss via dual regulation of bone formation and inflammatory cytokines. (A) Representative micro-CT images of distal femora (top) with three-dimensional reconstruction of the region of interest (bottom), with the blue indicating higher-density bone and the red indicating lower-density bone. (B – D) Statistical quantification of trabecular bone microstructural parameters (BMD, BV/TV, and Tb.Th). Inter-group comparisons were analyzed by one-way ANOVA. (E) Representative images of trabecular bone area in distal femur sections stained with hematoxylin and eosin. Scale bar, 200 μm or 50 μm. (F) Quantitative analysis of the trabecular bone area of the distal femur stained with hematoxylin and eosin. Inter-group comparisons were analyzed by one-way ANOVA. (G) Immunohistochemical staining of OCN in distal femurs. Scale bar, 200 μm or 50 μm. (H) Quantitative analysis of OCN-positive area. Inter-group comparisons were analyzed by one-way ANOVA. (I) Western blotting analysis of inflammatory cytokine expression (IL-6, TNF-α, and IL-1β) in mouse bone tissues. (J) Inflammatory cytokine expression (IL-6, TNF-α, and IL-1β) in mouse serum was detected by ELISA. Inter-group comparisons were analyzed by one-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; “ns” indicates non-significant differences.

Article Snippet: We purchased the Mouse P1NP (Procollagen 1 N-Terminal Propeptide) ELISA Kit (Cat#e-el-m0233), Mouse IL-6 ELISA Kit (Cat#e-el-m0044), Mouse TNF-α ELISA Kit (Cat#e-el-m3063), and Mouse IL-1β ELISA Kit (Cat#e-el-m0037) from Elabscience (Wuhan, China).

Techniques: Micro-CT, Staining, Immunohistochemical staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Genes & Diseases

Article Title: Targeting MDK alleviates bone loss via dual regulation of osteogenic differentiation and inflammatory cytokine expression

doi: 10.1016/j.gendis.2025.101931

Figure Lengend Snippet: Recombinant MDK protein triggers the activation of inflammatory cytokines through the NF-κB signaling pathway. (A, B) IL-6, TNFα, and IL-1β expression levels were detected using Western blotting. MC3T3-E1 cells were treated with recombinant MDK protein (600 ng/mL). Osteogenic differentiation was induced for 7 days. Inter-group comparisons were analyzed by a two-tailed unpaired Student's t -test (for normally distributed data with equal variance). (C, D) Western blotting analysis of NF-κB signaling pathway molecules in MC3T3-E1 cells treated with recombinant MDK protein for 7 days during osteoblastic differentiation. Inter-group comparisons were analyzed by two-tailed unpaired Student's t -test (for normally distributed data with equal variance). (E, F) IL-6 and IL-1β expression levels were detected using Western blotting. MC3T3-E1 cells were pretreated with 10 μM BAY 11–7082. Osteogenic differentiation was induced for 7 days. Inter-group comparisons were analyzed by one-way ANOVA. ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: We purchased the Mouse P1NP (Procollagen 1 N-Terminal Propeptide) ELISA Kit (Cat#e-el-m0233), Mouse IL-6 ELISA Kit (Cat#e-el-m0044), Mouse TNF-α ELISA Kit (Cat#e-el-m3063), and Mouse IL-1β ELISA Kit (Cat#e-el-m0037) from Elabscience (Wuhan, China).

Techniques: Recombinant, Activation Assay, Expressing, Western Blot, Two Tailed Test

Journal: Genes & Diseases

Article Title: Targeting MDK alleviates bone loss via dual regulation of osteogenic differentiation and inflammatory cytokine expression

doi: 10.1016/j.gendis.2025.101931

Figure Lengend Snippet: Schematic representation of MDK alleviating bone loss. MDK is significantly elevated in the serum of postmenopausal osteoporotic women and ovariectomized mice. Due to estrogen deficiency, iMDK alleviates bone loss by promoting bone formation and inhibiting inflammatory factors. Recombinant MDK protein inhibits osteogenic differentiation through the PI3K/AKT signaling pathway and up-regulates inflammatory factors IL-6, TNF-α, and IL-1β via the NF-κB signaling pathway.

Article Snippet: We purchased the Mouse P1NP (Procollagen 1 N-Terminal Propeptide) ELISA Kit (Cat#e-el-m0233), Mouse IL-6 ELISA Kit (Cat#e-el-m0044), Mouse TNF-α ELISA Kit (Cat#e-el-m3063), and Mouse IL-1β ELISA Kit (Cat#e-el-m0037) from Elabscience (Wuhan, China).

Techniques: Recombinant