Journal: bioRxiv

Article Title: Multi-stromal organoid co-cultures model pancreatic cancer and pancreatitis epithelial cell-fibroblast heterogeneity

doi: 10.64898/2025.12.05.692494

Figure Lengend Snippet: (A) Bright field images of organoids generated from normal pancreas (hereon, termed N), pancreatitis (hereon, termed P) and PDAC tissues after > 5 passages; scale bars = 50 μm. (B) Bright field images (taken with an Incucyte organoid module) of N, P and PDAC organoids at day 3 and day 5 post-plating, as part of a growth assay; scale bars = 800 μm. (C) Summary table of the N, P and PDAC organoid lines analysed by RNA-sequencing (RNA-seq) after 3 days of culture in complete media. (D) Principal component analysis (PCA) of N (n=5 biological replicates), P (n=5 biological replicates, each with 2 technical replicates at different passages) and PDAC (n=4 biological replicates, of which one with 2 technical replicates at different passages) organoids, as assessed by RNA-seq. (E) RNA-seq expression of ductal ( Sox9 , Krt19 , Pdx1 ), acinar ( Ptf1a , Cpa1 ), endocrine ( Ins2 , Chga ), immune ( Ptprc ), endothelial ( Pecam1 ) and fibroblast ( Col1a1 ) markers in N, P and PDAC organoids. Results show mean ± SEM. (F) Heatmap showing Gene Set Variation Analysis enrichment (GSVA) scores (averaged per group) of selected pathways in N, P and PDAC organoids, as assessed by RNA-seq. Pathways displayed correspond to pathways significantly upregulated in GSEA in P organoids ( and/or 3F). (G) Western blot of phospho-STAT3 (p-STAT3) and STAT3 in P and PDAC organoids cultured in complete media for 3 days. ACTIN, loading control. (H) GSEA of the murine pancreatitis Ductal 1 (left) and Ductal 2 (right) signatures in PDAC organoids compared to P organoids. The signatures were significantly downregulated in PDAC organoids (i.e. significantly enriched in P organoids). The signatures were obtained by comparing pancreatitis Ductal 1 and Ductal 2 cells, respectively, to PDAC malignant cells from the murine snRNA-seq dataset. NES, normalized enrichment score. FDR, false discovery rate. (I) GSEA of the PDAC malignant signature in PDAC organoids compared to P organoids. The signature was obtained by comparing PDAC malignant cells to pancreatitis ductal cells from the murine snRNA-seq dataset. The signature was significantly enriched in PDAC organoids.

Article Snippet: Organoid proliferation was followed for 120 hours with an Incucyte organoid module (Sartorius) with measurement of the organoid area per well every 3 hours (4 technical replicates per measurement).

Techniques: Generated, Growth Assay, RNA Sequencing, Expressing, Western Blot, Cell Culture, Control

Journal: Communications Biology

Article Title: HuB serves as the central hub for connecting HuR-related mRNAs with translation machinery in inflammation

doi: 10.1038/s42003-025-09228-9

Figure Lengend Snippet: The expression of HuR, HuB, HuC and HuD in various cell lines and mice lungs. HEK293, HeLa, RAW264.7 and U251 cells were treated with TNFα or LPS for 1 h. Subsequently, Western blotting was performed to detect the protein levels of HuR, HuB, HuC and HuD ( A ). Mice lungs were challenged with LPS (1 mg/kg) for 1 h. Then lung homogenates were prepared, Western blotting was performed to detect the protein levels of HuR, HuB, HuC and HuD ( B ). Quantification of the relative levels of HuR and HuB from the western blotting was achieved by normalization against that of β-actin. C Immunofluorescence analysis of the localization and expression of HuB in mice lungs tissue under inflammatory conditions. Mice lungs were challenged with LPS (1 mg/kg) for 1 h, Then frozen sections of mice lungs were prepared. Expression of HuB was detected by HuB antibody. Nuclei were counterstained with DAPI. Scale bar, 50 μm. D The Hel-N1 isoform of HuB is mainly expressed by brain tissues. Total RNA was extracted from various tissues and cells as indicated, and the purified RNA from each sample was transcribed to cDNA, and the HNS domain of HuB was amplified, The PCR products were sent for sequencing, and the information was compared with the NCBI database to identify the isoforms of HuB (Hel-N1 or Nel-N2). Shown is the diagram of the sequences of HuB mRNAs from the different tissues and cells. E Inflammation stimulation enhances the association of HuB with HuR. HEK293 cells were transfected with GFP-HuB plasmid, and then mock-treated or exposed to TNFα (10 ng/mL) for 1 h. The cytosolic lysates were collected for co-IP assay by using GFP antibody, and then subjected to western blotting to detect the interaction of GFP-HuB with HuR. F ITC binding measurements of GST-HuR with GST-HuB. The purified recombinant proteins (GST and GST-HuR) were injected into calorimetric cells before GST-HuB injection. Theoretical titration curves as well as associated KD values were displayed. G , H Time dynamics of HuR shuttles to the cytoplasm in response to inflammatory stimulation. HEK293 cells were challenged with TNFα (10 ng/mL) for different time intervals, the cells were lysed as cytosolic extracts (CE) and nuclear extracts (NE), and the cytoplasmic location of HuR was analyzed by western blotting ( G ) and immunefluorescence staining (H) with HuR antibody. The profiles in the right panel ( H ) show the fluorescence intensity of HuR from line scans in the merged images in the left panel, analyzed by Image Pro Plus software (red line: HuR; blue line: DAPI). Scale bar, 20 μm. I Knockdown of HuB reduces the TNFα-induced cytoplasmic shuttling of HuR. HEK293 cells were transfected with siRNA targeting HuB or a control. After being mock-treated or exposed to TNFα (10 ng/mL) for 1 h, the CE and NE of cells were prepared, the efficacy of HuB knockdown was showed by western blotting with HuB antibody, and the shuttling of HuR was detected by western blotting with HuR antibody. J The ectopic over-expression of HuB but not HuR increases the cytoplasmic location of HuR. Flag-HuR or Flag-HuB together with GFP-HuR were overexpressed in HEK293 cells. The location of GFP-HuR was visualized with a *60 objective on a confocal microscope. Scale bar, 20 μm. The quantification from randomly three independent experiments was shown in the right panel. Quantification shows mean ± SD based on three independent experiments, with significance of the difference determined by one-way ANOVA ( A , B , E , J ). n = 3 animals ( B , C ), n = 3 independent experiments ( A , D – J ), * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance. Source data are available in supplementary data 1 for this figure.

Article Snippet: Curve fitting to a single binding site model was performed using the ITC data analysis module of MicroCal Software provided by the manufacturer.

Techniques: Expressing, Western Blot, Immunofluorescence, Purification, Amplification, Sequencing, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Binding Assay, Recombinant, Injection, Titration, Staining, Fluorescence, Software, Knockdown, Control, Over Expression, Microscopy