human primary colonic fibroblasts  (ATCC)

Journal: bioRxiv

Article Title: TWEAK is increased in ulcerative colitis and contributes to fibroblast-mediated monocyte activation via heterologous non-canonical NF-kB/STAT3 signalling

doi: 10.1101/2025.09.23.678006

Figure Lengend Snippet: (A) Venn diagrams comparing the DEG upregulated and downregulated in THP1s co-cultured (CC) with Vehicle or TWEAK-treated FBLs compared to naïve THP1s. (B) Heat map of selected genes dysregulated by co-culture with FBLs (n=3, >1.5 fold, p<0.05). (C) (D) Pathway enrichment analysis of transcription factors represented by genes differentially upregulated in THP1s co-cultured with TWEAK-treated fibroblasts. (E-F) Immunoblot analysis and quantification of pSTAT3/STAT3 in THP1s (E) and primary monocytes (F) cultured with conditioned media from human primary colonic fibroblasts (FBL CM) untreated or treated with TWEAK for 48 hours.

Article Snippet: Human primary colonic fibroblasts (CCD-18CO, American Type Culture Collection (ATCC), Manassas, VA) were cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC) supplemented with 10% Foetal bovine serum (FBS, BioSciences, Ireland).

Techniques: Cell Culture, Co-Culture Assay, Western Blot

Journal: bioRxiv

Article Title: TWEAK is increased in ulcerative colitis and contributes to fibroblast-mediated monocyte activation via heterologous non-canonical NF-kB/STAT3 signalling

doi: 10.1101/2025.09.23.678006

Figure Lengend Snippet: (A) Schematic of the indirect co-culture method. Primary colonic fibroblasts were treated with TWEAK alone or TWEAK+NIK inhibitor for 48 hours, and their conditioned medium used to stimulate THP1s for 24 hours. (B) Immunoblotting analysis of STAT3 phosphorylation in THP1 cells stimulated with conditioned medium from fibroblasts treated with TWEAK alone or in the presence of the NIK inhibitor NIK SMI01. (C) Relative pSTAT3/STAT3 ratio quantified from (B) via densitometry analysis. (D-E) Relative expression (mRNA) of NOD2 , ICAM1 , IL12 , OSM , and IL1B in THP1s stimulated with conditioned medium from fibroblasts treated with TWEAK alone or in the presence of the NIK inhibitor, quantified by qPCR. (F) Immunoblotting analysis and quantification of IL1B expression at protein level in THP1s stimulated with conditioned medium from fibroblasts.

Article Snippet: Human primary colonic fibroblasts (CCD-18CO, American Type Culture Collection (ATCC), Manassas, VA) were cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC) supplemented with 10% Foetal bovine serum (FBS, BioSciences, Ireland).

Techniques: Co-Culture Assay, Western Blot, Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: TWEAK is increased in ulcerative colitis and contributes to fibroblast-mediated monocyte activation via heterologous non-canonical NF-kB/STAT3 signalling

doi: 10.1101/2025.09.23.678006

Figure Lengend Snippet: (A) Flow cytometry analysis of CD90 + /PDPN + stromal cells (CD45 - /EpCAM - ) isolated from the colonic mucosa of healthy donors or UC patients with either active disease (inflamed and non-involved sites) or in remission. (B) Quantification of the frequency of CD90 + /PDPN + stromal cells in the previous biopsies (normalised to 50k stromal cells). (C) Correlation between the frequency of CD90 + /PDPN + stromal cells and the abundance of TWEAK + myeloid cells. (D) UMAP showing the different stromal cell clusters identified by scRNAseq (reanalysed from Smillie et al 2019 ) (E) Expression of Fn14 (TNFRSF12A), OSMR and IL1R1 within the stroma in healthy and inflamed UC mucosa. Reanalysed from Smillie et al 2019 . (F) Dot plot showing the level (colour) and percentage (size) of expression of the TWEAK receptor (Fn14), Podoplanin (PDPN), and key components of the non-canonical NF-κB signalling pathway. (G) Histograms showing the expression of Fn14 by flow cytometry in naive colonic fibroblasts and colonic fibroblasts treated with 5 ng/ml TNF for 24h. (H) Quantification of the mean fluorescence intensity (geometric mean) from (F). ** p<0.01, *** p<0.001, **** p<0.0001, n≥4.

Article Snippet: Human primary colonic fibroblasts (CCD-18CO, American Type Culture Collection (ATCC), Manassas, VA) were cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC) supplemented with 10% Foetal bovine serum (FBS, BioSciences, Ireland).

Techniques: Flow Cytometry, Isolation, Expressing, Fluorescence

Journal: bioRxiv

Article Title: TWEAK is increased in ulcerative colitis and contributes to fibroblast-mediated monocyte activation via heterologous non-canonical NF-kB/STAT3 signalling

doi: 10.1101/2025.09.23.678006

Figure Lengend Snippet: (A) Immunofluorescence staining of inflammatory fibroblasts (PDPN, Magenta) and monocytes (CD14, cyan) in FFPE colonoscopy biopsies from UC patients. Detail panel shows co-occurrence between fibroblasts and monocytes (yellow arrows). (B) Quantification of PDPN expression by mean fluorescence intensity from (A). (C) Quantification of the average number of monocytes in the vicinity of fibroblasts within a field of view in (A). (D) Correlation between the frequency of CD90 + /PDPN + stromal cells and monocytes (CD45 + /CD11b + /CD14 + ) in biopsies from UC patients and healthy donors. ** p<0.01, n≥5.

Article Snippet: Human primary colonic fibroblasts (CCD-18CO, American Type Culture Collection (ATCC), Manassas, VA) were cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC) supplemented with 10% Foetal bovine serum (FBS, BioSciences, Ireland).

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Single-cell analysis identifies tumour-associated fibroblasts as source expressing PAI-1 (A–C) UMAP plots showing composition of cell types in peritoneal tumors and ascites derived from colorectal and ovarian cancer. (D and E) SERPINE1 (gene encoding PAI-1), along with other fibroblast-specific genes, were significantly expressed in fibroblasts but not in other cell types for both the colorectal and ovarian cancer cases. (F) Within the peritoneal nodules and ascites samples examined, SERPINE1 is significantly expressed in fibroblasts over other cell types. Values represent p values adjusted for multiple hypotheses using the Bonferroni correction. (G) PAI-1 secretion in stromal (mesothelial cells and fibroblasts) cell lines, measured in conditioned medium at 24 h by ELISA. CCD-18Co is a normal colonic fibroblast cell line, CAF05 is a colorectal tumor cancer-associated fibroblast cell line, and HM-3/TERT and LP9-TERT are normal peritoneal mesothelial cell lines. PAI-1 secretion in PC cell lines (SNU-C1 and Colo-205) were below the detection limit and not represented in the figure. (G) is representative of three independent biological experiments. Graph shows mean ± SEM. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, unpaired two-sided t test.

Article Snippet: CCD-18Co, human normal colonic fibroblast, was purchased from ATCC and cultured in EMEM with 10% FBS.

Techniques: Single-cell Analysis, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay