Journal: bioRxiv

Article Title: Fibroblasts sense spatial proximity via an EGFR–CREB5 axis to restore quiescent synovial lining in remission rheumatoid arthritis

doi: 10.64898/2025.12.10.693501

Figure Lengend Snippet: Bulk RNA-seq of CREB5 KD in patient-derived synovial fibroblasts reveals CREB5-dependent integration of cell density in fibroblast lineage programs. A. Representative immunoblots of pCREB5 (T61) and total CREB5 in synovial fibroblasts after CREB5 knockdown, cultured at low (20 cells/mm²) or high (100 cells/mm²) density, with GAPDH as loading control (top). Densitometric quantification of pCREB5 (T61) and total CREB5 normalized to GAPDH (bottom). B. PCA of the normalized gene expression values after batch correction for individual cell line variability. Each point represents the expression profile of one sample. C. GO terms enrichment analysis showing the functional pathways associated with genes upregulated or downregulated in response to CREB5 KD across different cell densities. D. Expression profiles of synovial lining markers ( PRG4 , PDPN , CLU ) and sublining markers ( POSTN , THBS1 , COL1A1 ) across four cell densities in control and CREB5 KD conditions. E. Fisher’s exact test showing the enrichment of AMP-defined lining genes among diffrerntially expressed genes after CREB5 KD. F. Immunoblot analysis of pCREB (S133) and total CREB5 in synovial fibroblasts cultured at 100 cells/mm² for 3 days and stimulated with forskolin (10 μM, 30 min) or DMSO (0.1%, control) prior to lysis, with β-actin as loading control. G. qRT-PCR analysis of lining markers in Synovial fibroblast cultured at at low (20 cells/mm²) and high (100 cells/mm²) density for 6 hours followed by stimulation with forskolin (7 μM) or 0.1% DMSO control for 72 hours. Data represent biological triplicates, and P values are indicated above the bars.

Article Snippet: Membranes were blocked for 15 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) then incubated overnight at 4°C with primary antibodies against CREB5 (Proteintech, #14196-1-AP, 1:500 dilution), p-CREB (Cell Signaling Technology, #9198, 1:500), EGFR (Proteintech, # 66455-1-Ig),p-EGFR (Cell Signaling Technology, #3777), p-ATF2(Cell Signaling Technology, # 24329, 1:300), SOX5 (Proteintech, #13216-1-AP, 1:500), FOXO1 (Proteintech, #18592-1-AP, 1:500), EGFR (Proteintech, #66455-1-Ig), GAPDH (Thermo Fisher Scientific, #MA5-15738), ɑ-tubulin (11224-1-AP), or beta-actin (Cell Signaling Technology, #3700).

Techniques: RNA Sequencing, Derivative Assay, Western Blot, Knockdown, Cell Culture, Control, Gene Expression, Expressing, Functional Assay, Lysis, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Fibroblasts sense spatial proximity via an EGFR–CREB5 axis to restore quiescent synovial lining in remission rheumatoid arthritis

doi: 10.64898/2025.12.10.693501

Figure Lengend Snippet: EGFR signaling regulates CREB5 activation and synovial fibroblast lineage identity in a cell density–dependent manner. A. Schematic diagram of the experimental design and timelines. B. UMAP representation of single-cell spatial transcriptomic profiles from synovial fibroblasts colored by condition (control vs. siRNA knockdown). C. UMAP plot of synovial fibroblasts grouped by density and EGFR KD condition. (Left) Cells colored by density state (high vs. low) highlight the separation of lining-like and sublining-like populations. (Right) Cells colored by experimental conditions showing control (high and low density) versus EGFR knockdown ( EGFR _high and EGFR _low). D. Heatmap showing UCell scores for low-density gene signatures across all knockdown conditions at high cell density (600 cells/mm²). Each column represents a different knockdown condition or control. The color gradient indicates the strength of a low-density UCell score. E. Violin plots showing the distribution of CLU and PDPN expression in synovial fibroblasts across control and EGFR KD conditions. The x-axis represents experimental conditions and the y-axis represents normalized gene expression. F. Representative immunoblots of total EGFR, pEGFR (Y1068), pCREB5 (T61), and total CREB5 in synovial fibroblasts following HB-EGF stimulation or EGFR KD, cultured at low (20 cells/mm²) or high (100 cells/mm²) density, with α-tubulin as loading control. G. Quantification of EGFR, pEGFR (Y1068), pCREB5 (T61), and total CREB5 immunoblots, normalized to α-tubulin as a loading control. H. qRT-PCR analysis of lining ( PRG4 and CLU ) and sublining marker ( CD90 and POSTN ) expression in synovial fibroblasts cultured at varying cell densities under control or EGFR KD conditions. Data represent biological triplicates, with P values indicated above the bars.

Article Snippet: Membranes were blocked for 15 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) then incubated overnight at 4°C with primary antibodies against CREB5 (Proteintech, #14196-1-AP, 1:500 dilution), p-CREB (Cell Signaling Technology, #9198, 1:500), EGFR (Proteintech, # 66455-1-Ig),p-EGFR (Cell Signaling Technology, #3777), p-ATF2(Cell Signaling Technology, # 24329, 1:300), SOX5 (Proteintech, #13216-1-AP, 1:500), FOXO1 (Proteintech, #18592-1-AP, 1:500), EGFR (Proteintech, #66455-1-Ig), GAPDH (Thermo Fisher Scientific, #MA5-15738), ɑ-tubulin (11224-1-AP), or beta-actin (Cell Signaling Technology, #3700).

Techniques: Activation Assay, Control, Knockdown, Expressing, Gene Expression, Western Blot, Cell Culture, Quantitative RT-PCR, Marker

Journal: bioRxiv

Article Title: Fibroblasts sense spatial proximity via an EGFR–CREB5 axis to restore quiescent synovial lining in remission rheumatoid arthritis

doi: 10.64898/2025.12.10.693501

Figure Lengend Snippet: HB-EGF–EGFR–CREB5 signaling drives density-dependent lining fibroblast differentiation. A. Representative immunoblots of pCREB5 (T61) and total CREB5 in synovial fibroblasts cultured at high density (100 cells/mm²) following stimulation with EGF, HB-EGF, or TGFα (100 ng/ml, 10 min) or no stimulation (control), with GAPDH as loading control. B. Quantification of pCREB5 (T61) and total CREB5 in immunoblots of ligand stimulation, normalized to GAPDH as a loading control. C. qRT-PCR analysis of lining ( PRG4 and CLU ) and sublining marker ( POSTN and COL1A1 ) expression in synovial fibroblasts cultured at low (20 cells/mm²) and high (100 cells/mm²) densities after stimulation for 72 hours with HB-EGF (100 ng/ml), or without stimulation (control). Data represent biological triplicates, with P values indicated above the bars. D. qRT-PCR analysis of lining ( PRG4 and CLU ) and sublining marker ( POSTN and COL1A1 ) expression in synovial fibroblasts cultured at low (20 cells/mm²) and high (100 cells/mm²) densities after HBEGF KD. Data represent biological triplicates, with P values indicated above the bars. E. Representative immunoblots of pCREB5 (T61) and total CREB5 in synovial fibroblasts cultured at low density (20 cells/mm²) or high density (100 cells/mm²) following HBEGF KD, F. Quantification of pCREB5 (T61) and total CREB5 in immunoblots of HBEGF KD, normalized to α-tubulin as a loading control. G. Spatial distribution of lining and sublining fibroblasts in RA patient tissue, with gene expression mapped for CREB5 , HBEGF , and EGFR . H. Model of density-dependent fibroblast differentiation through HB-EGF/EGFR-CREB5 signaling

Article Snippet: Membranes were blocked for 15 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) then incubated overnight at 4°C with primary antibodies against CREB5 (Proteintech, #14196-1-AP, 1:500 dilution), p-CREB (Cell Signaling Technology, #9198, 1:500), EGFR (Proteintech, # 66455-1-Ig),p-EGFR (Cell Signaling Technology, #3777), p-ATF2(Cell Signaling Technology, # 24329, 1:300), SOX5 (Proteintech, #13216-1-AP, 1:500), FOXO1 (Proteintech, #18592-1-AP, 1:500), EGFR (Proteintech, #66455-1-Ig), GAPDH (Thermo Fisher Scientific, #MA5-15738), ɑ-tubulin (11224-1-AP), or beta-actin (Cell Signaling Technology, #3700).

Techniques: Western Blot, Cell Culture, Control, Quantitative RT-PCR, Marker, Expressing, Gene Expression

Journal: Journal of Animal Science and Biotechnology

Article Title: Unique Sertoli cell adaptations support enhanced spermatogenesis in chickens

doi: 10.1186/s40104-025-01304-8

Figure Lengend Snippet: CREB5 played a crucial role in the maturation of chicken Sertoli cells. A Statistical analysis of the number of differentially expressed genes between mature and immature Sertoli cells. Red represents the number of upregulated genes, and blue represents the number of downregulated genes. B A dot bubble chart showing representative GO terms enriched in differentially expressed genes. C Expression patterns of representative genes of mature and immature Sertoli cells. D A dot bubble chart showing representative KEGG pathways enriched in differentially expressed genes. E A chord plot showing the subordinate relationship between representative genes (Left) and KEGG pathways (Right). The color of the boxes in front of the gene names from blue to red represents the fold change in gene expression. F Schematic of Sertoli cell extraction and CREB5 interference. G and H The mRNA and protein expression of CREB5. I and J The mRNA and protein expression of ZO-1, and occludin in chicken Sertoli cells ( n = 3). K The mRNA expression of AR , LAMA5 , NOTCH2 in chicken Sertoli cells ( n = 3). * P < 0.05, ** P < 0.01

Article Snippet: After blocked with 5% non-fat milk for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies, including CREB5 (Proteintech, Cat# 14196-1-AP), NPAS2 (Santa cruz, Cat# sc-134404), RORα (Proteintech, Cat# 10616-1-AP), ZO-1 (Proteintech, Cat# 21773-1-AP), occludin (Proteintech, Cat# 27260-1-AP), STAR (OmnimAbs, Cat# OM153513 ), HSD3B1 (abcam, Cat# ab65156), CYP11A1 (CellSignalingTechnology, Cat# 14217).

Techniques: Expressing, Gene Expression, Extraction

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Characterizing 24-Hour Skeletal Muscle Gene Expression Alongside Metabolic and Endocrine Responses Under Diurnal Conditions

doi: 10.1210/clinem/dgae350

Figure Lengend Snippet: Gene expression assay targets in human skeletal muscle ( vastus lateralis )

Article Snippet: CREB5 , cAMP responsive element binding protein 5 , Hs00191719_m1.

Techniques: Gene Expression, Binding Assay