il11  (Miltenyi Biotec)

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: After stimulation, fibroblasts were detached with trypsin, washed in PBS, and re-suspended in live cell sorting buffer (PBS with 5% FBS, 25mM HEPES, 1mM EDTA) and the top and bottom 15% of IL11 mNG fibroblasts were sorted on a Sony SH800 cell sorter (see Extended Data Fig. 6a for gating strategy).

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: After stimulation, fibroblasts were detached with trypsin, washed in PBS, and re-suspended in live cell sorting buffer (PBS with 5% FBS, 25mM HEPES, 1mM EDTA) and the top and bottom 15% of IL11 mNG fibroblasts were sorted on a Sony SH800 cell sorter (see Extended Data Fig. 6a for gating strategy).

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: After stimulation, fibroblasts were detached with trypsin, washed in PBS, and re-suspended in live cell sorting buffer (PBS with 5% FBS, 25mM HEPES, 1mM EDTA) and the top and bottom 15% of IL11 mNG fibroblasts were sorted on a Sony SH800 cell sorter (see Extended Data Fig. 6a for gating strategy).

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) Masson’s trichrome stained Glis3 f/f and Glis3 f/f ;Cre mouse colons (5–18 weeks) treated with water or chronic DSS (left). Total collagen percentage quantification across distinct biological replicates from four pooled experiments (right). Glis3 f/f -water, n=18; Glis3 f/f ;Cre -water, n=21; Glis3 f/f -DSS, n=20; Glis3 f/f ;Cre -DSS, n=15 mice. (b) Quantification of colonic hydroxyproline normalized to total protein from tissue lysates from (a). (c) qPCR quantification of collagens normalized to Eef2 from tissue lysates from (a). (d) Colon length measurements from (a). (e) Histopathological scoring (see Methods ) on H&E-stained tissues from (a). (f) Top: Xenium-based spatial profiling schematic of water- or chronic DSS-treated Glis3 f/f and Glis3 f/f ;Cre mice. Bottom: distribution of cell type proportions across water- or chronic DSS-treated Glis3 f/f and Glis3 f/f ;Cre mice. n=3 mice per condition. (g) Spatial projection of mIAFs and activated macrophages on colonic Swiss-rolls of water- or chronic DSS-treated Glis3 f/f and Glis3 f/f ;Cre mice. (h) Spatial projection of the GLIS3 signature module score on colonic Swiss-rolls of water- or chronic DSS-treated Glis3 f/f and Glis3 f/f ;Cre mice. (i) Dot plot showing Il11, Glis3, and the GLIS3 signature expression on each replicate of the Xenium spatial sequencing cohort. n=3 mice per condition. (j) Dot plot showing highlighted pro-inflammatory gene expression from activated macrophages and neutrophils in water- or chronic DSS-treated Glis3 f/f and Glis3 f/f ;Cre mice. n=3 mice per condition. All mice were co-housed and models of chronic DSS followed the same regimen (2.0%, 42 days). Images are representative of the sample cohort. 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: After stimulation, fibroblasts were detached with trypsin, washed in PBS, and re-suspended in live cell sorting buffer (PBS with 5% FBS, 25mM HEPES, 1mM EDTA) and the top and bottom 15% of IL11 mNG fibroblasts were sorted on a Sony SH800 cell sorter (see Extended Data Fig. 6a for gating strategy).

Techniques: Staining, Expressing, Sequencing, Gene Expression, 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 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: After stimulation, fibroblasts were detached with trypsin, washed in PBS, and re-suspended in live cell sorting buffer (PBS with 5% FBS, 25mM HEPES, 1mM EDTA) and the top and bottom 15% of IL11 mNG fibroblasts were sorted on a Sony SH800 cell sorter (see Extended Data Fig. 6a for gating strategy).

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) 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: After stimulation, fibroblasts were detached with trypsin, washed in PBS, and re-suspended in live cell sorting buffer (PBS with 5% FBS, 25mM HEPES, 1mM EDTA) and the top and bottom 15% of IL11 mNG fibroblasts were sorted on a Sony SH800 cell sorter (see Extended Data Fig. 6a for gating strategy).

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: After stimulation, fibroblasts were detached with trypsin, washed in PBS, and re-suspended in live cell sorting buffer (PBS with 5% FBS, 25mM HEPES, 1mM EDTA) and the top and bottom 15% of IL11 mNG fibroblasts were sorted on a Sony SH800 cell sorter (see Extended Data Fig. 6a for gating strategy).

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: After stimulation, fibroblasts were detached with trypsin, washed in PBS, and re-suspended in live cell sorting buffer (PBS with 5% FBS, 25mM HEPES, 1mM EDTA) and the top and bottom 15% of IL11 mNG fibroblasts were sorted on a Sony SH800 cell sorter (see Extended Data Fig. 6a for gating strategy).

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

Journal: International Journal of Molecular Sciences

Article Title: Extended PRF: Impact of Heat on Gene Expression in Gingival Fibroblasts

doi: 10.3390/ijms26189120

Figure Lengend Snippet: PPP, buffy coat, and e-PRF induced a substantial increase of IL11 in gingival fibroblasts, showing expression as x-fold increases compared with untreated cells. ( A ) Real-time PCR analysis of IL11. The expression level of the untreated cells was set to 1 for calibration. ( B ) Quantification of IL11 levels in the supernatant by immunoassay. Statistical analysis was based on the ratio-paired t -test. Wo stood for the untreated cells. N = 4, and different symbol shapes indicate independent experiments. Significance was set at p < 0.05.

Article Snippet: The immunoassay for human IL11 (DY218, R&D Systems, Minneapolis, MN, USA) was performed with the supernatant of gingival fibroblasts exposed to the lysates of PPP, heated PPP (Alb-gel), buffy coat (BC), and e-PRF.

Techniques: Expressing, Real-time Polymerase Chain Reaction