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: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet: A. Schematic of CRISPR activation system. B. Histograms showing H2-K1 and CD47 staining in B16-dCas9 cells. C. Tumor growth over time for ICB treated mice for H2-K1 asgRNA tumors (red) relative to control (gray) (on left) and Cd47 asgRNA B16-dCas9 tumors (red) relative to control (gray) (on right). D. Schematic of in vivo pooled gain-of-function screen. E. Volcano plot of primary CRISPR activation screen hits. Genes marked in color are known immune regulators or novel hits of interest. Depleted targets of interest are marked in green and enriched targets of interest are marked in orange and Creb5 is marked in Red to highlight the top enriched gene across both screens. F. Schematic for secondary screen gene selection. G. Volcano plot of secondary CRISPR activation screen hits. Genes marked in color are known immune regulators or novel hits of interest. Depleted targets of interest are marked in green and enriched targets of interest are marked in orange and Creb5 is marked in Red to highlight the top enriched gene across both screens.

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques: CRISPR, Activation Assay, Staining, Control, In Vivo, Selection

Journal: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet: A. Creb5 transcript and protein expression in B16 control and Creb5 asgRNA cells. B.CREB5 protein expression in nucleus (purple color) of cancer cells from tumor tissue. C. Schematic of in vivo competition assay. D. Results of in vivo competition assay showing log2 fold change for Creb5 asgRNA relative to average of two control asgRNAs in B16 tumors. E-F. Tumor growth over time of E. Creb5 asgRNA tumors (red) relative to control (gray) in ICB treated mice and F. Creb5 ORF tumors (red) relative to control (gray) in ICB treated mice. G. Schematic of in vivo competition assay. H. Results of in vivo competition assay showing log2 fold change for Creb5 asgRNA2 relative to control in B16 tumors. I. Kaplan-Meier curve showing progression-free survival for melanoma patients treated with anti-PD-1 with either high (red) or low (grey) CREB5 expression. Low and high cohorts are separated by median CREB5 expression. P-value from log-rank test. J. Cox proportional-hazards model evaluating the relationship between tumor purity, CREB5 expression, and metastatic stage (mStage). All variables are z-scored prior to fitting the model. 95% confidence interval indicated. K. CREB5 expression (TPM) in tumors from anti-PD-1-treated patients categorized as either responders (grey) or progressors (red). Difference in means calculated by Mann-Whitney U test.

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques: Expressing, Control, In Vivo, Competitive Binding Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet: A. B16 Tumor growth over time in NSG (Black), WT (Grey), WT + ICB (Red) mice. B. Results of in vivo competition assay showing log2 fold change for Creb5 asgRNA relative to control in B16 tumors. C. Tumor growth over time of Creb5 asgRNA tumors (Red) relative to control (Gray) in untreated mice. D. Survival curve for Creb5 asgRNA tumors (Red) relative to control (Gray) in ICB treated mice. E. CREB5 protein expression in B16 cells expressing control or CREB5 ORF. F. Tumor growth over time in untreated mice and G. Survival curve in ICB treated mice for CREB5 ORF tumors (red) relative to control (Gray). H. B16 Tumor growth over time in NSG (Black), WT (Grey), WT + ICB (Red), and WT + ICB + aCD8 (Purple) mice. I. In vivo competition assay showing log2 fold change for Creb5 asgRNA1 relative to control in B16 tumors. J. Creb5 transcript abundance in B16 and YUMMER cells K. Creb5 transcript abundance in YUMMER control Creb5 KO cells (Blue) relative to control (Gray) and protein expression. L. Results of in vivo competition assay showing log2 fold change for Creb5 KO sgRNA relative to control in YUMMER tumors. M. Creb5 transcript abundance in YUMMER Creb5 asgRNA cells (Red) relative to control (Gray). N. Results of in vivo competition assay showing log2 fold change for Creb5 asgRNA relative to control in YUMMER tumors.

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques: In Vivo, Competitive Binding Assay, Control, Expressing

Journal: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet: A. Heatmap showing hierarchical clustering and significantly differentially expressed genes between Creb5 OE or control B16 cells. B. Bar graph showing the top 6 enriched and depleted gene sets in Creb5 asgRNA cells relative to control B16 cells. C-D. Mountain plot showing enrichment score for the C. Hallmark EMT gene set and D. Mesenchymal-like gene set in Creb5 asgRNA and control B16 cells. Collagen and collagen-related genes are called out. E. Transcript abundance for collagen and collagen-stabilizing factors in Creb5 OE (top) or Creb5 KO (bottom) B16 cells relative to control. F. Protein expression of collagen genes in B16 control and Creb5 ORF cells. G. Immunofluorescence (IF) staining for nuclei (DAPI) and Collagen 1A1 from control (Top) or Creb5 OE (Bottom) tumors. H. Quantification of IF staining. I. Bar graph showing the top 6 gene sets positively and negatively correlated with CREB5 expression in malignant cells from scRNA-seq of human patient melanoma tumors. J-K. Mountain plot showing enrichment score for the J. EMT gene set and K. Mesenchymal-like gene set. Genes are ranked based on their correlation with CREB5 expression in malignant cells from scRNA-seq of human patient melanoma tumors. Collagen and collagen-related genes are called out. L. Volcano plot showing the correlation of all transcription factors with the Mesenchymal-like gene signature. GSEA was used to quantify this relationship. Known regulators of the mesenchymal state are red. M. Bar graph showing the top 6 gene sets positively and negatively correlated with CREB5 expression from bulk tumor RNA-seq. N. Mountain plot showing enrichment score for the Mesenchymal-like gene set. Genes are ranked based on their correlation with CREB5 expression. O. Bar graph showing the top 6 gene sets positively and negatively correlated with progression-free survival when controlling for variation in tumor purity and metastatic stage. P. Mountain plot showing enrichment score for the Mesenchymal-like gene set. Genes are ranked based on their association with PFS calculated from Cox PH model controlling for tumor purity and metastatic stage.

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques: Control, Expressing, Immunofluorescence, Staining, RNA Sequencing

Journal: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet: A. PCA of transcriptional profiling of Creb5 asg1, Creb5 asg2, and control B16 cells. The top genes driving variation between the samples are plotted as vectors. B. Bar graph showing the gene set pathways enriched in Creb5 asg1 cells relative to control B16 cells. C-D. Mountain plot showing enrichment score for the C. EMT gene set and D. Mesenchymal-like gene set in Creb5 asg1 cells relative to control B16 cells. Gene symbols and ranks for collagen and collagen-related genes are marked in red. E. Transcript abundance for collagen and collagen-stabilizing factors in Creb5 asgRNA cells (Red, top) or Creb5 sgRNA cells (Blue, bottom) relative to control (Grey) in YUMMER cells. F. Transcript abundance of FOXA1 motifs in the promoter region of Col1a1 , Col4a1 , and Col16a1 . G. Zoom out IF image (500um) of control OE tumors (Left) and Creb5 OE tumors (Right) shown in . H. Representative images of trichrome staining in control OE (Top), Creb5 OE (Middle) and Col1a1 OE (Bottom) tumors. I. Col1a1 IF staining of control OE (Top), Creb5 OE (Middle) and Col1a1 OE (Bottom) tumors and quantification on left. J. Transcript abundance for collagen and collagen-stabilizing factors in Creb5 asgRNA cells (Red, top) or Creb5 sgRNA cells (Blue, bottom) relative to control (Grey) in A375 cells. K. Bar graph showing the gene set pathways correlated with CREB5 expression in human melanoma patient samples. L-M. Mountain plot showing enrichment score for the H. EMT gene set and I. Mesenchymal-like gene set for correlation with CREB5 expression in human melanoma patient samples. Gene symbols and ranks for collagen and collagen-related genes are marked in red.

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques: Control, Staining, Expressing

Journal: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques:

Journal: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet: A. Transcript abundance of Col1a1 (Left), Col4a1 (Center), Col16a1 (Right) in B16 cells with respective target asgRNA (Red) relative to control (Grey). B. COL4A1 IHC staining on control OE (Left) and Creb5 OE tumors (Center), and quantification of pixel coverage over area (Right). C. B16 Tumor growth over time of Col1a1 asg (Left), Col4a1 asg (Center) and Col16a1 asg (Right) in red relative to control asg in gray in WT mice. D. Transcript abundance of Loxl1 in B16 Loxl1 KO sgRNA cells (Blue) relative to control (Gray). E. IF staining of COL1A1 (Red), CD31 (White), and DAPI (Blue) in control ko tumors (Left) and Loxl1 ko tumors (Center) and quantification of pixel coverage of COL1A1 per area (Right, top) and per cell (Right, bottom). F. Transcript abundance of Loxl1 in B16 Loxl1 KO sgRNA cells (Blue) relative to control (Gray) on left and, transcript abundance of Loxl2 in B16 Loxl2 KO sgRNA cells (Blue) relative to control (Grey) on Right. G. Schematic of in vivo competition assay. H. Results of in vivo competition assay showing log2 fold change for Creb5 asgRNA relative to control in B16 control KO tumors (Left) or B16 Loxl1 KO tumors (Center) and B16 Loxl2 ko (Right).

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques: Control, Immunohistochemistry, Staining, In Vivo, Competitive Binding Assay

Journal: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet: A-D. Tumor growth over time in ICB treated mice for A. Col1a1 OE tumors (Red) relative to control (Gray), B. Col4a1 OE tumors (Red) relative to control (Gray), and C. Col16a1 OE tumors (Red) relative to control (Gray). D. Schematic of LOXL-mediated collagen crosslinking. E. Schematic of in vivo competition assay. F. Results of in vivo competition assay showing log2 fold change for Creb5 asgRNA relative to control in B16 control KO tumors (Left) or B16 Loxl1 KO tumors (Right). G-I. Tumor growth over time in ICB treated mice for G. Creb5 OE (Red) in Loxl1 -deficient (Blue) or Loxl1 -sufficient (Red) tumors relative to control OE in Loxl1 -deficient (Gray) or Loxl1 -sufficient (Black) tumors. H. Col1a1 OE tumors with Loxl1 -deficient (Blue) or Loxl1 -sufficient (Red) tumors relative to control OE in Loxl1 -deficient (Gray) or Loxl1 -sufficient (Black) tumors. I. Col4a1 OE tumors with Loxl1 -deficient (Blue) or Loxl1 -sufficient (Red) tumors relative to control OE in Loxl1 -deficient (Gray) or Loxl1 -sufficient (Black) tumors.

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques: Control, In Vivo, Competitive Binding Assay

Journal: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet: A-E. Representative images of Multiplex Immunofluorescence (IF) of paraffin embedded formalin fixed B16 tumors sections post ICB treatment with CD45, COL1A1, CD8, F4/80, CD4, FOXP3 and DAPI. All of the IF images depict the same section in which IF was performed simultaneously but analyzed in different channels. A. Zoom out picture of Control-OE tumor on top and Creb5 -OE tumor on bottom, showing CD45 (White), Dapi (Blue), Col1a1 (Pink), with marked tumor edge and tumor center for subsequent zoom in images. B-E. Zoom in images of Control-OE tumors (Top) and Creb5 -OE tumor (Bottom) with section of tumor center (Left) and tumor edge (Right) and quantification of total pixel per total number of cells. B. CD45 (White), Dapi (Blue), and COL1A1 (Pink). C. F4/80 (White), Dapi (Blue), and COL1A1 (Pink). D. CD8 (White), Dapi (Blue), and COL1A1 (Pink). E. CD4 (Red), FOXP3 (Yellow), Dapi (Blue), and COL1A1 (Pink). F-O. Flow cytometry analysis of TILs isolated from B16 tumors post ICB treatment. Dot plot showing control OE tumors (Left), Creb5 OE tumors (Center), and quantification of percentage positive cells (Right). F. Dot plot showing CD45 staining on tumor cells post digestion. G. Dot plot showing TCRb and NK1.1 stains on cells post lymphocyte enrichment and gated on CD45 positive population on left and quantification of TCRb positive T cells, NK1.1 positive NK cells and TCRb and NK1.1 negative myeloid cells as percentage of total CD45 positive cells in control OE (Gray) and Creb5 OE (Red) tumors on right. H. Dot plot showing CD11b and F4/80 stains in CD45 positive population on left and quantification of CD11b and F4/80 double positive macrophages as percentage of total CD45 positive cells in control OE (Gray) and Creb5 OE (Red) tumors on right. I. Dot plot showing CD11b and Gr-1 stains in CD45 positive on left and quantification of CD11b and Gr-1 double positive MDSCs as percentage of total CD45 positive cells in control OE (Gray) and Creb5 OE (Red) tumors on right. J. Dot plot showing CD11b and MHC-II stains in CD45 positive population on left and quantification of CD11b and MHC-II double positive DCs as percentage of total CD45 positive cells in control OE (Gray) and Creb5 OE (Red) tumors on right. K. Dot plot showing TCRb and CD8 stains in CD45 positive population on left and quantification of TCRb and CD8 double positive T cells as percentage of total CD45 positive cells in control OE (Gray) and Creb5 OE (Red) tumors on right. L. Dot plot showing Foxp3 stain in CD45, TCRb and CD4 positive population on left and quantification of Foxp3 negative T-helper cells and Foxp3 positive T regulatory cells as percentage of total CD4 T cells in control OE (Gray) and Creb5 OE (Red) tumors on right. M. Histograms showing LAIR1 staining on CD4 positive cells (yellow), NK (Brown), CD8 (Red) and CD11b positive myeloid cells (Deep red). N. Histograms showing LAIR1 staining on CD45 positive cells and quantification of LAIR1 MFI on CD45 positive cells in control OE (Gray) and Creb5 OE (Red) tumors on the right. O. Quantification of LAIR1 MFI on immune cells in control OE (Gray) and Creb5 OE (Red) tumors on the right.

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques: Multiplex Assay, Immunofluorescence, Control, Flow Cytometry, Isolation, Staining

Journal: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet: A. Tumor growth over time in ICB treated mice for Creb5 OE tumors (Red) relative to control (Gray).

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques: Control

Journal: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet: A. Schematic of in vivo competition assay. B. Results of in vivo competition assay showing log2 fold change for Creb5 asgRNA (Left), Col16a1 asgRNA (Middle), and Col17a1 asgRNA (Right) relative to control in B16 tumors. C. Schematic of collagen LAIR1 signaling. D. Transcript abundance for human LAIR2 in Control (Left) or LAIR2 ORF B16 cells. E. Schematic of in vivo competition assay. F. Results of in vivo competition assay showing log2 fold change for Creb5 asgRNA relative to control in B16 Control ORF tumors (Left) or B16 LAIR2 ORF tumors (Right). G. Schematic of PDOTs culture. H. Histogram plot showing the LAIR1 staining in patients tumor infiltrating lymphocytes. I. Representative IF images of PDOTs with no treatment or aPD-1 treatment (Top) or treated with recombinant human LAIR2 (2ug/ml) alone or in combination with aPD-1 (Bottom). J. Quantification of PDOTs IF images.

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques: In Vivo, Competitive Binding Assay, Control, Staining, Recombinant

Journal: bioRxiv

Article Title: CREB5 promotes immunotherapy resistance via tumor-intrinsic collagen matrix deposition

doi: 10.1101/2025.04.22.649109

Figure Lengend Snippet: A. Results of in vivo competition assay showing log2 fold change for Creb5 asgRNA1 (Left), Col16a1 asgRNA3 (Middle), and Col17a1 asgRNA4 (Right) relative to control in B16 tumors. B. Transcript abundance of CREB5 in A375 and patient samples.

Article Snippet: The following primary antibodies were used for detecting designated protein expression in western blot assays: CREB5 (Novus Biologicals, H00009586-M02, Abnova), CREB5 (ThermoFisher, PA-565593, Invitrogen), Anti-Mouse GapDH (Abcam, ab8245), Anti-Rabbit GapDH (Abcam, ab9485), COL1A1 (ThermoFisher, PA5-29569), COL4A1 (ThermoFisher, PA5-104508), COL16A1 (ThermoFisher, PA5-103382).

Techniques: In Vivo, Competitive Binding Assay, Control

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