Journal: Nature Communications

Article Title: Epithelial NF-κB shapes protective Treg cell responses to restrain intestinal inflammation

doi: 10.1038/s41467-026-78796-z

Figure Lengend Snippet: a Medium from freshly isolated organoids analysed by proteome array. n = 4 mice per group, pooled. Analysis: one-way ANOVA with Tukey’s correction. In bold: known NF-κB target genes. b Treg cell migration transwell assay: isolated MLN/ spleen primary Treg cells from Wt mice incubated with factors shown. NC (negative control – medium alone) or PC (positive control – Fetal bovine serum). Analysis: one-way ANOVA; Bonferroni’s multiple comparison test. Mean and SD shown. n = 9. c IHC against CCL22 ( n = 5 mice). Black triangles: CCL22+ immune cells. Purple triangles: CCL22+ epithelial cells. Scale: 10 μm. d Primary Treg cells from Wt mice cultured with organoid conditioned medium from Wt vs CCL22 −/− mice ( n = 3 mice), treated with LPS (25 μg/ml LPS over 48 h) or left untreated. Treg cell migration quantified via Transwell migration assay. Analysis: one-way ANOVA. Mean and SD shown. e Treg cell co-culture with conditioned organoid medium (SN), 30 Vol.-% and/or + 30 ng/mL murine IL-33, CM + 30 ng/mL IL-33 or CM + 100 ng/mL Amphiregulin. The fold change increase of expanded Treg cells on day 3 was measured in relation to the initial cell count. n = 3 mice. Analysis: as in a. Mean and SD shown. f Quantitation of CCR4+ and FoxP3+ cells from IF per image. n = 9. Mean and SD shown. g Isolated Wt ( n = 5) and IκBα IEC KO ( n = 3) CD4 + CD25+ Treg cells cultured for 6 h in medium supplemented with 50 ng/mL PMA and 1 µg/mL ionomycin. Supernatants analysed by ELISA. Analysis: unpaired t-test, two-sided. Mean and SD shown. h scRNA of colonic versus splenic immune cells from Wt and IκBα IEC KO mice. i Genes with significant changes in expression between colonic or splenic Treg cells in Wt versus IκBα IEC KO mice. j Differential expression of Gdpd3 and Ftl1 in spleen and colon Treg cells. Analysis: two-sided Wilcoxon rank-sum test k Colonic IF against GDPD3, CD4 and CDH1. Scale: 50 μm. n = 12. Zoom: double positive cells. l Quantitation of GDPD3+CD4+ cells per image. Analysis as in ( a ). Mean and SD shown. m Primary Treg cells from Wt mice cultured with organoid SN (supernatants). n = 3 mice. GDPD3+ cells visualized by IF. Analysis: as in ( a ). Mean and SD shown. Source data are provided as a Source Data file.

Article Snippet: 4.5 × 10 4 cells/well were seeded in RPMI medium without additives on a transwell insert (8μm pore size, Greiner Bio-One Cat. 662638) in a 24- well plate.

Techniques: Isolation, Organoids, Analysis, Migration, Transwell Assay, Incubation, Negative Control, Positive Control, Comparison, Cell Culture, Transwell Migration Assay, Co-culture Assay, Cell Characterization, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative Proteomics

Journal: Nature Communications

Article Title: Epithelial NF-κB shapes protective Treg cell responses to restrain intestinal inflammation

doi: 10.1038/s41467-026-78796-z

Figure Lengend Snippet: a scRNA dataset ( GSE116222 ) is based on samples from inflamed and non-inflamed colon regions of UC patients, as well as those from healthy controls. Visualization of IEC cells across the three conditions. b TRRUST analysis of top upregulated TF networks across the three conditions (Healthy, UC and UC NI (non-inflamed)) in IEC presented as a volcano plot. Text in blue: factors belonging to the NF-κB axis. A Gene-set enrichment was performed using Enrichr. Enrichment significance was assessed using Fisher’s exact test, and enrichment magnitude was represented by the odds ratio. Volcano plots generated with the Enrichr Appyter display odds ratio versus −log 10 ( P value). c TRRUST analysis of downregulated TF networks in UC-inflamed colon IEC versus healthy IEC. Text in blue: factors belonging to the NF-κB axis. Analysis as in ( b ). d H&E of colon sections from healthy and UC patients. Scale: 50 μm. e Colitis score (Nancy index) from ( d ). The patient number is shown in the graph. Analysis: Two-tailed t -test. Mean and SD shown. f Colon-derived organoids were derived from colon biopsies of UC patients or non-IBD controls, with at least n = 4 patients per group. Growth of colon organoids was measured over several days. Mean and SD shown. Analysis: t- test, two-tailed, unpaired. g At least 10 colon organoids per patient from healthy ( n = 3 persons) and UC colon-derived organoids ( n = 4 patients) treated with LPS over 48 h and stained with antibodies as indicated. Scale: 50 μm. h Quantification from g. Top panel: Ki67 (not significant). Lower panel: cleaved caspase 3. Analysis: One-way ANOVA with Tukey’s correction. Mean and SD shown. i Colon organoids were treated with TNF (TNF was removed, and the medium was changed) or left untreated. Medium from colon-derived organoids was analysed by proteome array, and the presence of secreted factors quantitated from bulk ( n = 4) samples per group and n = 2 technical replicates. Significance determined by one-way ANOVA with Tukey’s correction. Only significant results are shown. j Analysis of proteome data by DAVID v2023q4 followed by REVIGO for visualization of GO terms on semantic space. Bubble size indicates prevalence. Color: orange—terms enriched in medium of UC colon-derived organoids treated with TNF, purple—terms enriched in medium of healthy colon-derived organoids treated with TNF. SD and mean shown. Analysis: Welch’s t -test, two-sided – no significance. k Transwell recruitment with Jurkat cells incubated in medium from colon-derived organoids from UC patients or healthy controls. Analysis: one-way ANOVA with Tukey’s correction. n = 4 for healthy samples and n = 6 for UC samples (pooled). Individual points indicate technical replicates. Mean and SD shown. Source data are provided as a Source Data file.

Article Snippet: 4.5 × 10 4 cells/well were seeded in RPMI medium without additives on a transwell insert (8μm pore size, Greiner Bio-One Cat. 662638) in a 24- well plate.

Techniques: Analysis, Generated, Two Tailed Test, Derivative Assay, Organoids, Staining, Incubation, Significance Assay

Journal: Nature Communications

Article Title: Cell-derived lactate triggers local hydrogelation and phenotype remodelling

doi: 10.1038/s41467-026-77702-x

Figure Lengend Snippet: Unless stated otherwise, data are mean ± s.d. Statistics: unpaired two-tailed Welch’s t -test; P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P > 0.05 (ns, not significant). a Transwell assay schematic: A549 cells in the lower well; AlgTA + lactate oxidase (LOx) + horseradish peroxidase (HRP) in the insert. Cell-secreted lactate diffuses into the insert and is oxidised by LOx to generate H 2 O 2 , which HRP uses to oxidatively couple AlgTA phenols and form a gel (see also Fig. ). Created in BioRender. Braun, M. (2026) https://BioRender.com/569s2eu . b Representative fluorescence micrographs of A549 cells at days 1–3. Stains: Calcein AM (live, green), NucBlue (nuclei, blue), propidium iodide (dead, red), n = 3 independent experiments. Scale bars, 100 µm. c Resazurin-based metabolic activity ( n = 9 wells) from 3 independent experiments. Shown P -values: day 1, P = 0.054, day 2, P < 0.001; day 3, P < 0.001. d Relative proteome profiling of selected tumour-associated proteins in AlgTA/LOx/HRP-treated cultures at day 2 versus matched controls ( n = 3 biological replicates, 10 wells each, 2 on-membrane replicates). Shades of red indicate decreased, and shades of green indicate increased relative protein abundance. e Evolution of mechanics and transport: Young’s modulus E measured by nanoindentation ( n = 9 indentations from 3 gels; see the “Methods” section) and fluorescence correlation spectroscopy (FCS) diffusion-time ratio ( τ gel / τ buffer ) for gels formed at days 1–3 after equilibration in tracer-containing PBS ( n = 4 positions per gel). f Lactate concentration under the indicated conditions at days 1–3. ( n = 6 samples from 2 independent experiments). Shown P -values: day 1, P = 0.005; day 2, P < 0.001; day 3, P < 0.001. g Extracellular H 2 O 2 quantified by a fluorimetric hydrogen peroxide assay at days 1–3 ( n = 4 samples).

Article Snippet: 25.000 A549 cells were seeded in a 24-well plate and cultured using 600 μL Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose, Gibco 31053-028) supplemented with 10% dialysed foetal bovine serum (FBS, Gibco A33820-01) and 1% Penicillin/Streptomycin for 24 h. The medium was exchanged, the gel precursor-filled Transwell (Greiner Bio-One 662641) was added, and the experiment started.

Techniques: Two Tailed Test, Transwell Assay, Fluorescence, Activity Assay, Profiling, Membrane, Quantitative Proteomics, Spectroscopy, Diffusion-based Assay, Concentration Assay, H2O2 Assay

Journal: Nature Communications

Article Title: Cell-derived lactate triggers local hydrogelation and phenotype remodelling

doi: 10.1038/s41467-026-77702-x

Figure Lengend Snippet: Unless stated otherwise, data are mean ± s.d. (day 2). Statistics: unpaired two-tailed Welch’s t -test; P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P > 0.05 (ns, not significant). Formulation: 3 wt% AlgTA, 10 U mL −1 HRP, 1 U mL −1 LOx; control: DMEM supplemented with FBS. a Communication cascade: spheroid-secreted lactate is converted by LOx/HRP to H 2 O 2 , driving phenol coupling and in situ AlgTA gelation around the spheroid (schematic) and photograph of encapsulated spheroids. Scale bar, 2 mm. Created in BioRender. Braun, M. (2026) https://BioRender.com/569s2eu . b Representative fluorescence micrographs of A549 spheroids: F-actin (phalloidin, green) and nuclei (NucBlue, blue), n = 3 independent experiments. Top: control (regular medium). Bottom: AlgTA/LOx/HRP, showing actin-rich protrusions. Scale bars, 100 µm. c Calibration of hydrogel crosslinking with defined lactate concentrations (green). Correlation between the diffusion-time ratio of Alexa488-BSA ( τ gel / τ buffer ) and known concentrations of lactate (0–10 mM sodium lactate). d Spatial transport mapping by FCS: diffusion-time ratio ( τ gel / τ buffer ) and apparent lactate-equivalent concentration ( c lac , eq ) versus distance from the spheroid surface. Individual values were taken at three parallel offset distances and are shown with a line representing the mean. e Proteomics (3D, day 2): relative abundance of selected tumour-associated proteins in AlgTA/LOx/HRP versus control ( n = 3 biological replicates, 12 spheroids each, 2 on-membrane replicates). Red indicates decreased, and green indicates increased relative protein abundance. f Proteomic profiling of treated AlgTA/LOx/HRP 2D Transwell and 3D spheroid cultures. Filled bars indicate the condition showing the stronger relative response. 3D: 3 biological replicates, 12 spheroids each, 2 on-membrane replicates; 2D: n = 3 biological replicates, 10 wells each, 2 on-membrane replicates. g Representative spheroid diameter with and without AlgTA/LOx/HRP; 16 spheroids per condition. Shown P -values: AlgTA/LOx/HRP day 0 vs day 2, P = 0.546; control day 0 vs day 2, P < 0.001; AlgTA/LOx/HRP day 2 vs control day 0, P < 0.001, n = 3 experiments. h Metabolism under encapsulation ( n = 3 independent experiments): CellTiter-Glo® 3D (ATP, n = 9 wells, P < 0.001) and resazurin (NADH, n = 9 wells, P < 0.001) assays.

Article Snippet: 25.000 A549 cells were seeded in a 24-well plate and cultured using 600 μL Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose, Gibco 31053-028) supplemented with 10% dialysed foetal bovine serum (FBS, Gibco A33820-01) and 1% Penicillin/Streptomycin for 24 h. The medium was exchanged, the gel precursor-filled Transwell (Greiner Bio-One 662641) was added, and the experiment started.

Techniques: Two Tailed Test, Formulation, Control, In Situ, Spheroids, Fluorescence, Diffusion-based Assay, Concentration Assay, Membrane, Quantitative Proteomics, Profiling, Encapsulation

Journal: Bioactive Materials

Article Title: Reconstructing the ischemic osteogenic microenvironment through hierarchical scaffolds orchestrating Mg 2+ signaling and neuropilin-1–mediated angiogenesis

doi: 10.1016/j.bioactmat.2026.02.031

Figure Lengend Snippet: Evaluation of angiogenic potential induced by Mg 2+ and NRP-1. ( A ) Scratch assay in three different microenvironments. ( B ) Transwell assay in three different microenvironments. ( C ) Quantitative analysis of wound healing area. ( D ) Quantitative analysis of number of migration cells. ( E ) Immunofluorescence staining of VEGFA and FGF2, with DAPI for nuclear staining and F-actin for cytoskeleton labeling. ( F ) Tube formation evaluation in four different microenvironments. ( G ) Quantitative analysis of number of junction. ( H ) Quantitative analysis of Flu intensity. ( I ) Western-blot analysis of VEGFA, FGF2, and Nr4a1. ( J-L ) Quantitative analysis of relative protein expression of VEGFA, FGF2, and Nr4a1. Data are presented as mean values ± s.d. (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (one-way ANOVA).

Article Snippet: For the Transwell assay, BMSCs were seeded in the upper chambers of Transwell inserts (8.0 μm pore size; Servicebio, China), while different culture conditions were applied in the lower chambers according to the experimental groups.

Techniques: Wound Healing Assay, Transwell Assay, Migration, Immunofluorescence, Staining, Labeling, Western Blot, Expressing