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: bioRxiv

Article Title: Long-range chromatin interactions on the inactive X and at Hox clusters are regulated by the non-canonical SMC protein Smchd1

doi: 10.1101/342212

Figure Lengend Snippet: a. Schematic of in vitro differentiation of mESC to neuromesodermal progenitors. mESC are derived in 2i + LIF medium. One week prior to differentiation (D-7) cells were infected with MSCV-Cre-puro or MSCV-puro retrovirus (green arrow), and transduced cells selected with puromycin (blue arrow). Cells were weaned into 15% Serum + LIF medium. For differentiation cells were changed into N2B27 medium containing Fgf-2. At day 2 (D2) Gsk3i, and at D6 Gdf11 were added. Cells were harvested for RNA-seq at day (D) 1, 5 and 8 (red arrows). b. Heatmaps showing the log 2 (fold change) expression differences in Hox genes between Smchd1 deleted and control samples at day 1, 5 and 8 of differentiation from mESC to neuromesodermal progenitors. Red to blue colour indicates fold change. Genes written in red have statistically significant differences at day 8, n=2. c. As for b , except samples are presomitic mesoderm from somite matched Smchd1 +/+ and Smchd1 MommeD1/MommeD1 E9.5 embryos. Somite numbers are indicated at the left. Log 2 fold change is shown for n=4 female Smchd1 MommeD1/MommeD1 and n=3 female Smchd1 +/+ embryos. d/f. Representative sagittal views of whole-mount E17.5 male Smchd1 +/+ ( d ) and Smchd1 MommeD1/MommeD1 ( f ) embryos with forelimbs and hindlimbs removed, stained with Alizarin red and Alcian blue for bone and cartilage, respectively. e/g. As per d/f showing dorsal view. Putative T13 is indicated with an asterix, note absence of rib on T13 in the Smchd1 MommeD1/MommeD1 embryo, representing homeotic transformation.

Article Snippet: After 48 hours (D2), medium was replaced with N2B27 medium supplemented with 10 ng/mL recombinant human basic FGF (Peprotech) and 5 μ M StemMACS CHIR99021 (Mitenyi Biotech).

Techniques: In Vitro, Derivative Assay, Infection, RNA Sequencing Assay, Expressing, Staining, Transformation Assay

Journal: bioRxiv

Article Title: Multi-species genome-wide CRISPR screens identify GPX4 as a conserved suppressor of cold-induced cell death

doi: 10.1101/2024.07.25.605098

Figure Lengend Snippet: a , Viability of hibernator cells (Syrian hamster embryonic fibroblasts, Greater horseshoe bat embryonic fibroblasts, and 13-lined ground squirrel embryonic fibroblasts) and non-hibernator cells (SV40-immortalized mouse embryonic fibroblasts, human adult kidney fibroblasts, human adult dermal fibroblasts, rat adult dermal fibroblasts, and mouse adult dermal fibroblasts) exposed to 4°C as measured by trypan blue staining (n = 4, **** P < 0.0001). b , Human kidney fibroblast GPX4 knockout cells exhibit reduced cold tolerance compared to WT cells. Left: Western blot of wild-type (WT) and GPX4 KO cells for GPX4 and β-actin loading control. Right: Viability of GPX4 KO cells is significantly lower than WT cells at 4°C as measured by trypan blue staining (n = 4, **** P < 0.0001). c , Gpx4 activity is essential for cold survival of primary human kidney fibroblasts. Human kidney fibroblasts were placed at 4°C and left untreated or treated with RSL3 (1 μM) and/or the ferroptosis inhibitor ferrostatin-1 (Fer-1, 1 μM) for 7 days (n = 4, **** P < 0.0001). d , Representative fluorescence images of human kidney fibroblasts after 4 days at 4°C with no treatment, 1 μM Fer-1, 1 μM RSL3, or 1 μM Fer-1 and 1 μM RSL3. Cultures were stained with Hoechst 33342 and propidium iodide (PI) to identify live cells. e , Gpx4 activity is essential for cold survival in hibernator cells (Syrian hamster embryonic fibroblasts, Greater horseshoe bat embryonic fibroblasts, and 13-lined ground squirrel embryonic fibroblasts) and non-hibernator cells (SV40-immortalized mouse embryonic fibroblasts, human adult kidney fibroblasts, human adult dermal fibroblasts, rat adult dermal fibroblasts, and mouse adult dermal fibroblasts). Cells were placed at 4°C and left untreated, treated with RSL3 (1 μM), and/or ferrostatin-1 (Fer-1, 1 μM) for 7 days (n = 4). f , Expanded Day 4 data from e) indicates that Gpx4 activity is essential for fibroblast survival in the cold across several hibernator and non-hibernator species. Cells were placed at 4°C and left untreated or treated with RSL3 (1 μM) and/or ferrostatin-1 (Fer-1, 1 μM) for 7 days (n = 4, **** P < 0.0001). g-h , Gpx4 activity is essential in g) mouse primary cortical neuron cultures and h) hamster primary cortical neuron cultures. Cells were placed at 4°C and left untreated, treated with RSL3 (1 μM) and/or the ferroptosis inhibitor ferrostatin-1 (Fer-1, 1 μM) for 1 or 4 days (n = 4). RSL3 treatment increased cell death, which was rescued by ferrostatin-1. All values show mean ± SEM, with significance measured by one-way ANOVA adjusted for multiple comparisons with Tukey’s HSD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns P > 0.05.

Article Snippet: Three hibernator (13-lined ground squirrel postnatal dermal fibroblasts [gift from Wei Li at Duke University], SV40-immortalized Greater horseshoe bat embryonic dermal fibroblasts [gift from Rudolf Jaenisch at the Whitehead Institute for Biomedical Research], and Syrian golden hamster embryonic primary dermal fibroblasts [isolated]) and five non-hibernator (mouse embryonic SV40-immortalized dermal fibroblasts [gift from Jonathan Weissman at the Whitehead Institute for Biomedical Research], adult human kidney fibroblasts [purchased from ATCC], adult human dermal fibroblasts [purchased from ATCC], adult rat dermal fibroblasts [purchased from ATCC], and adult mouse fibroblasts [purchased from ATCC]) primary cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific #12430054) supplemented with 10% fetal bovine serum (GeminiBio #100-106) and 1% penicillin/streptomycin (Thermo Fisher Scientific #15140122).

Techniques: Staining, Knock-Out, Western Blot, Control, Activity Assay, Fluorescence