human htert immortalized foreskin fibroblast bj 5ta cells  (ATCC)

Journal: bioRxiv

Article Title: Eukaryotic Initiation Factor 5B (eIF5B)-Driven Translational Control Impacts Oral Squamous Cell Carcinoma Pathophysiology

doi: 10.64898/2026.01.30.702967

Figure Lengend Snippet: BJ-5ta cells were transfected with either a non-specific control siRNA (siC) or an eIF5B-specific siRNA pool (si-eIF5B). (A) Following 24 h of transfection, cells were treated with either vehicle control or TRAIL (100 ng/mL). Cell viability was assessed after an additional 72 h using alamarBlue TM assays. (B) Annexin V and 7-AAD staining and flow cytometric analysis of transfected cells. The top two panels, siC and siC + TRAIL, the bottom two panels, si5B and si5B + TRAIL, were harvested at 96 h post-transfection and treated with Annexin V + 7AAD. Contour plots are divided into quadrants Live (lower left; cells staining negative for both 7-AAD and annexin V), Q1 (upper left; cells staining positive for 7-AAD but negative for annexin V), Early apoptosis (lower right; staining annexin V positive and 7-AAD negative), and Late apoptosis (upper right; staining positive for both 7-AAD and annexin V). (C) Representative immunoblots probing for eIF5B, XIAP, Bcl-xL, cIAP1, cFLIPs, DR 4, DR 5, and β-actin (internal control) are shown. ( D) Control and eIF5B-depleted BJ-5ta cells were treated with the 4EGI-1 inhibitor to inhibit cap-dependent translation. Levels of XIAP were monitored under control and the 4EGI-1 treatment conditions. (E) eIF5B-bound RNA immunoprecipitations (RIPs) followed by qRT-PCR measurements of input-normalized tRNAiMet levels from BJ-5ta vs UMSCC-29. Data are presented as mean ± SEM from three independent biological replicates, with statistical significance indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: CAL-33 and BJ-5ta (immortalized fibroblast) cell lines were obtained from American Type Culture Collection (ATCC; Virginia, USA), and UMSCC-1 and UMSCC-29 were obtained from the University of Michigan (Michigan, USA).

Techniques: Transfection, Control, Staining, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Eukaryotic Initiation Factor 5B (eIF5B)-Driven Translational Control Impacts Oral Squamous Cell Carcinoma Pathophysiology

doi: 10.64898/2026.01.30.702967

Figure Lengend Snippet: BrdU incorporation assay was used to measure cell proliferation. Quantification shows the percentage of BrdU-labeled cells in control (siC) and si5B-treated groups. Data are presented as mean ± SEM; ***p < 0.001. (A) UMSCC-29 (B) BJ-5ta (C) Collagen-based invasion assay was used to evaluate cell invasiveness in control (siC) and si5B-treated groups. Cells were seeded onto collagen matrices and allowed to invade for a defined period (48-72 hours). The number of invading cells was quantified and expressed as the mean ± SEM. Microscopy images of invading cells (C; left panel) , quantification (C; right panel) . Phase contrast microscopy images showing wound area in siC/si5B-treated OSCC cells at the time of scratch introduction (T = 0 h) & at T = 30 h wound closure (c; top panel) . Quantification of the percentage of wound area covered after 30 hours, normalized to wound area at the time of scratch introduction (c; lower panel) . (E) Representative images of the endothelial HUVEC cells tube formation assay treated with conditioned media from siC- or si5B-transfected OSCC. ImageJ angiogenesis macro software quantified Junctions, segments, and nodes. OSCC cells were transfected with either control siRNA (siC) or eIF5B-specific siRNA (si5B) and incubated for 96 hours. (F) The conditioned media for the control and eIF5B-depleted OSCC cells were analyzed for angiogenic biomarkers (lower panel) using EVE Technologies. (G) Representative images of immunoblots quantified probing for eIF5B, p-EGFR, EGFR, p-ERK, ERK, p-NF-kB, NF-kB, HIF1α, VEGFA, and β-actin (internal control). Data are presented as mean ± SEM from three independent biological replicates, with statistical significance indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001, and ****, p< 0.0001.

Article Snippet: CAL-33 and BJ-5ta (immortalized fibroblast) cell lines were obtained from American Type Culture Collection (ATCC; Virginia, USA), and UMSCC-1 and UMSCC-29 were obtained from the University of Michigan (Michigan, USA).

Techniques: BrdU Incorporation Assay, Labeling, Control, Invasion Assay, Microscopy, Tube Formation Assay, Transfection, Software, Incubation, Western Blot

Journal: bioRxiv

Article Title: Eukaryotic Initiation Factor 5B (eIF5B)-Driven Translational Control Impacts Oral Squamous Cell Carcinoma Pathophysiology

doi: 10.64898/2026.01.30.702967

Figure Lengend Snippet: BJ-5ta cells were transfected with either control siRNA (siC) or eIF5B-specific siRNA (si5B) and incubated for 96 hours. TRAIL (100 ng/mL) treatment was performed for 4 hours, then the media was collected. The conditioned media for the control and eIF5B-depleted BT5Ta cells were analyzed for angiogenic biomarkers using EVE Technologies. The level of distinct angiogenic biomarkers was decreased in the spent media of eIF5B-depleted BJ-5ta cells. Data are expressed as mean ± SEM for three biological replicates. Statistical significance is indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: CAL-33 and BJ-5ta (immortalized fibroblast) cell lines were obtained from American Type Culture Collection (ATCC; Virginia, USA), and UMSCC-1 and UMSCC-29 were obtained from the University of Michigan (Michigan, USA).

Techniques: Transfection, Control, Incubation

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Schematic of the integrated IBD atlas workflow that utilizes scRNA-seq or Xenium to profile non-IBD, CD, and UC patients. IBD: inflammatory bowel disease; CD: Crohn’s disease; UC: ulcerative colitis. (b) Proportion changes of fibroblasts stratified across disease. Box plots represent the quartiles with medians as the center, and whiskers the 10–90% range. Statistical analysis was performed using scCODA (Bayesian Dirichlet-multinomial model) with SMC as reference (FDR < 20%) ( Methods ). Number of samples for each category: non-IBD=29; CD inflamed=28; CD non-inflamed=54; UC inflamed=25; UC non-inflamed=22. (c) Pseudobulk expression heatmap of scaled average IAF-specific genes (Wilcoxon signed rank test [two-sided], p < 0.05; log fold change > 3; expression in > 25% of IAFs, < 10% of non-IAFs). IAF CSGs: inflammation-associated fibroblast cell-specific genes. (d) Pseudobulk scaled expression heatmap of IAF genes involved in extracellular matrix (ECM) deposition/organization or cytokine/chemokine production. (e) Dot plot showing effect size ( β ) and absolute log 2 fold change (|LogFC|) for niche enrichment across CD and UC compared to non-IBD. Blue indicates enrichment in non-IBD and red in CD and UC. Analysis performed using scCODA with N3 reference niche. FDR < 20% for niche change in abundance. (f) Heatmap of statistically enriched cell type proportion abundance across niches. Chi-squared test with p < 0.05 was set as the significance threshold. (g) Left: visualization of cellular niches projected onto a Xenium-profiled UC patient tissue section. Right: distribution of IAFs on the same tissue section, showing dense distribution in niche N1. (h) H&E section of UC tissue from (g) showing annotated anatomical and pathological tissue regions. Images are representative of the sample cohort. n=16 patients. (i) Heatmap depicting the enriched niches within anatomical and pathological tissue domains.

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Masson’s trichrome-stained Il11 f/f and Il11 f/f ;Cre colons (8–18 weeks) treated with water or chronic DSS (left). Total collagen percentage from three pooled experiments (right). Il11 f/f -water , n=10; Il11 f/f ;Cre -water, n=14; Il11 f/f -DSS , n=17; Il11 f/f ;Cre -DSS, n=10 mice. (b) Colonic hydroxyproline normalized to total protein from tissues from (a). (c) qPCR quantification of collagens normalized to Eef2 from tissues from (a). (d) Colon length measurements from (a). (e) Percentage of IL-11 mNG cells across lineages after indicated treatments. Water-treated, n=2; DSS-treated, n=3 mice. (f) Masson’s trichrome- (left) and immunofluorescence- (right) stained Il11 mNG tissues after DSS. Images are representative of 3 independent experiments. (g) Schematic of PDGFRA+ fibroblast isolation from acute and chronic DSS-treated Il11 mNG mice (8–14 weeks) (left). Dot plot mapping human fibroblast gene signatures across mouse fibroblasts (right). (h) Pseudobulk expression heatmap depicting scaled average expression of Il11 and mNeonGreen from acute and chronic DSS. (i) Spatial niche-aware probability of intercellular communication. Edge thickness or node size depicts communication strength. Significant signals received by IAFs (left) and sent from activated macrophages (right). (j) Immunofluorescence of chronic DSS-treated colons from Il11 mNG mice depicting proximal macrophage (CD68, red) and IL-11 mNG fibroblast (green) localization. Arrows indicate signal adjacency. Images are representative of 3 independent experiments. (k) Spatial projection of IAFs and activated macrophages in non-IBD and CD tissues. (l) Dot plot of IAF IL11 expression as a function of proximity to activated macrophages. (m) Secreted IL-11 measured from co-cultures of polarized primary human monocyte-derived macrophages, removed of agonists, with colonic fibroblasts for 24 hours. Fibroblasts only, n=4; fibroblasts+macrophages, n=2; fibroblasts+polarized macrophages, n=3 cell lines. Mice were co-housed and DSS followed the same regimen: acute (2.0%, 7 days), chronic (2.0%, 42 days). Unless otherwise stated, statistics are by a two-way ANOVA with Tukey’s multiple comparison test on distinct biological replicates and error bars are mean ± s.e.m. ns, not significant.

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Staining, Immunofluorescence, Isolation, Expressing, Derivative Assay, Comparison

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Volcano plots of enriched hits based on fold change (FC) enrichment and p values. CRISPRko, CRISPR knockout; CRISPRa, CRISPR activation. One-sided hypergeometric test. Multiple comparisons adjusted by FDR. (b) Pathway diagram of enriched hits from CRISPRko (red), CRISPRa (blue), or both screens (green) in known pathways. (c) Scatter plot of CRISPRko and CRISPRa screens’ shared hits. Statistically significant hits ( p < 0.05) are boxed and in black, and selected hits labeled. One-sided hypergeometric test. Multiple comparisons adjusted by FDR. (d) Heatmap depicting log fold change (FC) expression of CRISPR screen hits across human fibroblasts. Hit selection filtered by differential expression in IAFs (Wilcoxon test, adjusted p < 0.01 [two-sided]; > 1% expression in IAFs). (e) scRNA-seq of primary human fibroblasts stimulated with TGF-β and IL-1β (10 ng/mL). UMAP of timepoint clusters (left) and IL11 expression (center). High IL11 expression in subclusters 6 and 10 (right). (f) Top-ranked mean correlation values between high IL11 -expressing subclusters 6 and 10 from (e) and shared CRISPR screen hits from (c) reveal top enrichment of GLIS3 , with p = 1.6x10 −13 in cluster 6, and p = 3.9x10 −9 in cluster 10. (g) Relative percentage of IL-11 mNG median fluorescence intensity (MFI) in GLIS3 -perturbed immortalized Cas9- or dCas9-VP64 fibroblasts compared to controls after TGF-β and IL-1β stimulation (24 hours). Control, n=4; CRISPRko/a, n=2 cell lines. (h) Secreted IL-11 from distinct biological replicates measured after co-culture of primary human colonic GLIS3 CRISPRko (left) or GLIS3 CRISPRa (right) fibroblasts with TLR2/6-activated monocyte-derived macrophages. Error bars are the mean ± s.e.m. ns, not significant. Two-way ANOVA with Tukey’s multiple-comparisons test. n=3 cell lines.

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Genome Wide, CRISPR, Knock-Out, Activation Assay, Labeling, Expressing, Selection, Quantitative Proteomics, Fluorescence, Control, Co-Culture Assay, Derivative Assay

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Top: Venn diagram of downregulated genes in GLIS3 CRISPRko or upregulated in GLIS3 CRISPRa fibroblasts after TGF-β and IL-1β stimulation (10 ng/mL, 24 hours). Significant genes with Benjamini & Hochberg adjusted p < 0.05 (Wald test, two-sided) were intersected to derive effector genes. Bottom: heatmap of average log fold change (FC) expression relative to controls. Displayed are intersecting effector genes; asterisks mark ChIP-seq peaks. n=3 per condition. (b) Schematic of ChIP-seq in IL11 mNG fibroblasts with GLIS3 3XFLAG knock-in after TGF-β and IL-1β stimulation (10 ng/mL, 24 hours) (left). Pie chart depicts GLIS3 3XFLAG IP peak distribution (right). (c) Gene ontology analysis of GLIS3 3XFLAG peaks at 24- versus 0-hours. Benjamini-Hochberg adjusted p values from hypergeometric test (one-sided). See Methods for more details. (d) ChIP-seq tracks upstream of the first exon of IL11 in IgG and GLIS3 3XFLAG IP samples, summed across all replicate samples. (e) Predicted TF binding motifs and their associated TFs enriched in all GLIS3 3XFLAG peaks. (f) ChIP-qPCR schematic of FOSL1 and TEAD1 IP in control or GLIS3 CRISPRko IL11 mNG fibroblasts after TGF-β and IL-1β stimulation (10 ng/mL, 24 hours) (left). Heatmaps depict Z-score fold enrichment for FOSL1 (center) or TEAD1 (right) targets across replicates. n=4 cell lines per condition. (g) Top: schematic of GLIS3 signature derivation and PROTECT cohort analysis. Bottom: CIBERSORT-estimated IAF and macrophage proportions across PROTECT samples stratified by Mayo score. Box plots represent the quartiles with medians as the center, and whiskers represent 1.5* inter-quartile range. Grey lines indicate mean GLIS3 ssGSEA score ± s.e.m. ** p < 0.001 (one-sided) from ordinal probit regression of Mayo scores with ssGSEA and cell proportions (n=226). See Methods for more details. (h) Heatmap of scaled average expression of refined GLIS3 signature across control and UC patients stratified by the combined Mayo score.

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, ChIP-sequencing, Knock-In, Binding Assay, ChIP-qPCR, Control

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) UMAP of epithelial, immune, and stromal cell compartments from profiling IBD patients with scRNA-seq. (b) UMAP of epithelial, lymphoid, myeloid, fibroblast, and stromal cell compartments from profiling IBD patients with Xenium-based spatial transcriptomics. (c) Dot plot showing effect size ( β ) and absolute log 2 fold change (logFC) for cell type enrichment across CD and UC compared to non-IBD controls. Blue indicates enrichment in non-IBD and red indicates enrichment in CD and UC. Analysis was done using scCODA with the SMC cells as the reference. Only cell types that passed the significance threshold (FDR < 20%) were considered changed in abundance. See Methods for more details. (d) Pathway enrichment analysis of IAF-specific genes. Benjamini-Hochberg adjusted p values from a hypergeometric test (one-sided) for enriched pathways were plotted. See Methods for details. (e) Pseudobulk scaled expression heatmap of anti-TNF resistance signature 21 plotted across all fibroblast subtypes in the integrated IBD atlas.

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Sum of cell counts of each niche across all patients profiled in the spatial atlas. (b) Barplot depicting relative proportion of each niche along the gastrointestinal tract. (c) Barplot depicting relative proportion of each niche across the 16 patients profiled. (d) Heatmap depicting the proportion of cell type abundance across ileal (n=3) or colonic (n=13) niches. Only statistically enriched cell types are plotted (Chi-squared two-sided test, Bonferroni adjusted p < 0.05). (e) Expression dot plot of selected immune genes from activated macrophages. (f) Pathway enrichment analysis of activated macrophage-specific genes. Benjamini-Hochberg adjusted p values from a hypergeometric test (one-sided) for enriched pathways were plotted. (g) Barplot depicting relative proportion of each niche across qualitative grades of tissue ulceration. (h) Barplot depicting relative proportion of each niche across qualitative grades of tissue fibrosis. (i) Barplot depicting relative proportion of each niche across qualitative grades of tissue inflammation. (j) Heatmap of cell type proportions across distinct anatomical and histopathological tissue domains as defined by a pathologist on all human patient tissues profiled. Only statistically enriched cell types are plotted (Chi-squared two-sided test, Bonferroni adjusted p < 0.05).

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Schematic of Il11 f/f mice generated by flanking exons 2-4 with LoxP for Cre-mediated excision. (b) qPCR quantification of Il11 from tissue lysates from Fig. 2a , normalized to Eef2 . (c) Schematic of mNeonGreen knock-in at the Il11 terminus with homology-directed repair. (d) Percentage of starting weight of mice from Fig. 2a (see Methods for treatment). Filled lines represent s.e.m. Two-way repeated measures ANOVA. (e) Histopathological scoring (see Methods ) on H&E-stained tissues from Fig. 2a . (f) Gating strategy to quantify IL-11 mNG after water or DSS treatment. Plots are representative of the sample cohort. Related to Fig. 2e , g . (g) UMAP of PDGFRA+ fibroblasts after acute or chronic DSS. (h) Pseudobulk scaled expression heatmap of human IAF genes across PDGFRA+ fibroblasts after acute or chronic DSS. (i) Pseudobulk scaled expression heatmap of human anti-TNF resistance genes across PDGFRA+ fibroblasts after acute or chronic DSS. (j) Proportion changes of PDGFRA+ fibroblasts across DSS models. Box plots represent the quartiles with medians as the center, and whiskers the 10-90% range. Number of samples for each category: Acute DSS=3; Chronic DSS=2. (k) Secreted IL-11 measured from co-cultures of ligand-activated monocyte-derived macrophages (see Methods for concentrations) and primary human colon fibroblasts. n=3 cell lines per condition. (l) qPCR quantification of IL11 from fibroblasts co-cultured with TLR2/6-activated macrophages over time, normalized to HPRT . Filled lines represent s.e.m. n=3, except at 12 hours where n=2 cell lines. All mice were co-housed. Unless otherwise stated, statistics are by a two-way ANOVA with Tukey’s multiple comparison test on distinct biological replicates and error bars are mean ± s.e.m. ns, not significant.

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Generated, Knock-In, Staining, Expressing, Derivative Assay, Cell Culture, Comparison

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Schematic of IAF activation inference. See Methods for more details. TF, transcription factor. (b) Left: heatmap of scaled cell type-averaged IAF TF activity (one-sided Wilcoxon, p < 0.01; mean activity difference > 0.75). Center: heatmap of pseudobulk IAF TF expression (Wilcoxon, p < 0.01). Right: ligand frequency ( >5 among top 10 NicheNet predicted regulators). (c) Dot plot of ligand/receptors driving activated macrophage and fibroblast communication. (d) Ingenuity upstream regulator analysis of IAF genes (agonists: Z-score > 0). (e) Secreted IL-11 measured from primary human colonic fibroblasts stimulated with agonists from (a) (10 ng/mL, 24 hours). n=3 per condition for all except TSLP where n=2 cell lines. One-way ANOVA with Dunnet’s multiple comparison test on distinct biological replicates and error bars are the mean ± s.e.m. ns, not significant. (f) Dot plot of RNA of macrophage or fibroblast markers and TGF-β and IL-1β ligands/receptors across myeloid and fibroblast subtypes. (g) UMAP of myeloid/lymphoid compartments alongside IL1B or TGFB1 expression.

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Activation Assay, Activity Assay, Expressing, Comparison

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Secreted IL-11 after co-culture of fibroblast knockouts for TGFB1 - and IL1B -related ligands or receptors with TLR2/6-activated macrophages (top) or knockouts in activated macrophage knockouts with fibroblasts (bottom). n=3 cell lines per condition. (b) Z-score heatmap of IAF genes in TGFBR1/2 , IL1R1 CRISPRko fibroblasts co-cultured with TLR2/6-activated macrophages, normalized to HPRT. n=3 cell lines per condition. (c) qPCR of gene knockouts normalized to HPRT . Macrophages: IL-4/IL-13 (10 ng/mL); FSL-1 (10 ng/mL) + ATP (5 mM). Fibroblasts: media. n=2 cell lines per condition. (d) Secreted TGF-β and IL-1β after co-culture of primary human macrophages and fibroblasts (24 hours) ( Methods ). n=3 cell lines per condition. (e) Secreted IL-11 from primary colonic fibroblasts stimulated with TGF-β and/or IL-1β (10 ng/mL, 24 hours). Dashed lines: additive or synergistic response ( Methods ). One-way ANOVA with Tukey’s multiple-comparisons test. n=3 cell lines per condition. (f) Left: immunofluorescence of Il11 mNG colons after intraperitoneal injection with IgG control or dual anti-TGF-β and anti-IL-1β antibodies (100 μL in PBS, 100 μg/mouse). Right: IL-11 mNG cell percentage in all DAPI-imaged cells from two pooled independent experiments. Mice (co-housed, 13-20 weeks) were treated with chronic DSS (2.0%, 35 days). n=7 mice per condition. (g) qPCR quantification of Il11 from lysates from (f) normalized to Eef2 . Kruskal-Wallis test with Dunn’s multiple-comparison test. (h) Total colonic collagen percentage quantification from (f). (i) Quantification of colonic hydroxyproline normalized to total protein from lysates from (f). (j) Percent starting weight of mice from (f). Filled lines represent s.e.m. Linear mixed-effects analysis with Dunnett’s multiple comparison test. (k) Histopathological scoring ( Methods ) of H&E-stained tissues from (f). (l) Colon length measurements from (f). Unless otherwise stated, statistics are by a one-way ANOVA with Dunnet’s multiple comparison test on distinct biological replicates and error bars are the mean ± s.e.m. ns, not significant.

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Co-Culture Assay, Cell Culture, Immunofluorescence, Injection, Control, Comparison, Staining

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Gating strategy to quantify IL11 mNG after TGF-β and IL-1β stimulation (10 ng/mL, 24 hours). (b) Dot plot of IL-11 determinants increased in expression during inflammation in CD and UC (Wilcoxon signed rank two-sided test, Benjamini-hochberg adjusted p < 0.05). (c) Pseudobulk scaled average expression heatmap of Il11 , mNeonGreen , and Glis3 from acute and chronic DSS-treated mice. (d) Left: immunofluorescence of dual-color IL11 mNG fibroblast-Thp-1 macrophage co-cultures with or without TLR2/6 activation. Right: nuclear GLIS3 MFI quantification. Lines represent the median. Two-tailed Mann–Whitney U test. Steady-state, n=156; TLR2/6 stimulation, n=186 individual cells. (e) qPCR measurement of GLIS3 from primary human colonic fibroblasts co-cultured with TLR2/6-activated monocyte-derived macrophages, normalized to HPRT . Filled lines represent s.e.m. n=3 cell lines. (f) Nuclear GLIS3 quantification in IL11 mNG fibroblasts with knock-in of GLIS3 3XFLAG stimulated with TGF-β and/or IL-1β (10 ng/mL). Filled lines represent s.e.m. One-way ANOVA with Dunnett’s multiple comparison test. n=4 wells of median values from 6,908-12,459 fibroblasts. (g) qPCR measurement of Glis3 from the sample cohort in Extended Data Fig. 5f , normalized to Eef2 . One-way ANOVA with Dunnett’s multiple comparison test. Statistics are on distinct biological replicates and error bars are the mean ± s.e.m. ns, not significant.

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Genome Wide, CRISPR, Expressing, Immunofluorescence, Activation Assay, Two Tailed Test, MANN-WHITNEY, Cell Culture, Derivative Assay, Knock-In, Comparison

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Left: immunofluorescence of control or GLIS3 CRISPRa fibroblasts at steady state, stained for COL6 and DAPI (nuclei). Right: COL6 MFI quantification. Lines represent the median. Two-tailed Mann–Whitney U test. Control, n=244; GLIS3 CRISPRa, n=198 individual cells. (b) ChIP-qPCR of IL11 DNA in GLIS3 3XFLAG knock-in fibroblasts stimulated with TGF-β and IL-1β (10 ng/mL, 24 hours). Unpaired Student’s t-test (two-sided). n=5 cell lines per condition. (c) qPCR measurement of IL11 in IL11 mNG fibroblasts treated with scrambled control or FRA1 siRNA stimulated with TGF-β and IL-1β (10 ng/mL, 24 hours), normalized to HPRT . Two-way ANOVA with Tukey’s multiple-comparisons test. n=3 cell lines per condition. (d) Z-score heatmap for relative fold change in gene expression for FOSL1 targets after stimulation of control or FRA1 knockdown fibroblasts with TGF-β and IL-1β (10 ng/mL, 24 hours) against control non-stimulated fibroblasts, normalized to HPRT . n=3 cell lines per condition. (e) qPCR of IL11 in IL11 mNG fibroblasts treated with scrambled control or TEAD1 , TEAD3 , or dual TEAD1 and TEAD3 siRNA stimulated for TGF-β and IL-1β (10 ng/mL, 24 hours), normalized to HPRT . Two-way ANOVA with Tukey’s multiple-comparisons test. n=3 cell lines per condition. (f) Z-score heatmap for relative fold change in gene expression for TEAD1 targets after stimulation of control or TEAD1 , TEAD3 , or dual TEAD1 and TEAD3 knockdown fibroblasts with TGF-β and IL-1β (10 ng/mL, 24 hours) against control non-stimulated fibroblasts, normalized to HPRT . n=3 cell lines per condition. Statistics are on distinct biological replicates and error bars are the mean ± s.e.m. ns, not significant.

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Control, Immunofluorescence, Staining, Two Tailed Test, MANN-WHITNEY, ChIP-qPCR, Knock-In, Gene Expression, Knockdown

Journal: Nature

Article Title: Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit

doi: 10.1038/s41586-025-09907-x

Figure Lengend Snippet: (a) Schematic of Glis3 f/f mice generated by flanking exon 3 with LoxP for Cre-mediated excision. (b) Percent of starting weight of Glis3 f/f and Glis3 f/f ; Cre mice from Fig. 5a . Filled lines represent s.e.m. Two-way repeated measures ANOVA. (c) UMAP of epithelial, immune, fibroblast, and stromal compartments in Xenium-based spatial profiling of water- and chronic DSS-treated Glis3 f/f and Glis3 f/f ; Cre mice .

Article Snippet: Primary colon fibroblasts (CRL-1459), and immortalized fibroblasts (CRL-4001) were obtained from the American Type Culture Collection (ATCC).

Techniques: Generated

Journal: Ultrasonics Sonochemistry

Article Title: From lab to pilot: sonochemical coating of PDDA-based polymer dots for scalable, wash-durable antibacterial textiles

doi: 10.1016/j.ultsonch.2025.107619

Figure Lengend Snippet: Live/Dead assay of keratinocytes (a, b) and fibroblasts (c, d) after exposure to PDs-coated and uncoated cotton fabrics, respectively. (e) Cell viability after exposure to PDs-coated and uncoated cotton fabrics (n = 3).

Article Snippet: Human fibroblast (ATCC-CRL-4001, BJ-5ta) and keratinocyte (HaCaT) cell lines, S. aureus (ATCC 25923), E. coli (ATCC 25922) and P. aeruginosa (ATCC 10145) were obtained from American Type Culture Collection (ATCC LGC Standards).

Techniques: Live Dead Assay