Journal: bioRxiv

Article Title: JUNB O-GlcNAcylation-mediated promoter accessibility of metabolic genes modulates distinct epithelial lineage in pulmonary fibrosis

doi: 10.1101/2024.05.27.594700

Figure Lengend Snippet: a, Reactome analysis of gene signatures in aberrant basal cells shows a strong inclination towards glucose-related metabolic pathways. b , Schematic representation of the cell isolation process from human distal lung tissue to generate patient-derived AOs. c , Representative immunofluorescence staining of 15 days AOs shows a pseudostratified epithelium with basal cells (KRT5+, green) externally, and differentiated club cells (SCGB1A1+, red) and goblet cells (MUC5B+, red) internally. Nuclei are stained with DAPI (blue). Scale bars are 20 µm (first row) and 50 µm (second row). d , Quantification of ciliated cell area indicates a significant decrease in cilia in IPF AOs from n = 6 IPF and n = 6 control AO donors (mean + s.e.m, * p < 0.05, unpaired t -test). e , Organoid size quantification over 15 days shows an increased proliferative capacity in IPF AOs (n = 4 IPF and n = 7 control AO donors, mean + s.e.m, **** p < 0.0001, unpaired t -test). f , Profibrotic biomarker MMP7 secretion is increased in IPF AOs (n = 5 IPF and n = 5 control AO donors, mean + s.e.m., *** p < 0.001, unpaired t -test). g , Representative immunofluorescence staining of AOs reveals aberrant basal cells (KRT5+(green)/KRT17+ (yellow)/COL1A1+ (red)). Nuclei are stained with DAPI (blue). Scale bars: 50 µm. IPF-stimuli further enriches this population after 7 days. h, i , Seahorse XF Mito Fuel Flex Test kit showed increased glucose ( h ) and decreased glutamine ( i ) dependency of IPF and IPF-stimulated AOs (n = 5 IPF and n = 5 control donors, mean + s.e.m., * p < 0.05, ** p < 0.01, ANOVA/ Tukey’s).

Article Snippet: Measurement of human MMP7 (Biotechne, #DY907) was performed according to manufacturer’s instructions.

Techniques: Cell Isolation, Derivative Assay, Immunofluorescence, Staining, Biomarker Assay

Journal: bioRxiv

Article Title: JUNB O-GlcNAcylation-mediated promoter accessibility of metabolic genes modulates distinct epithelial lineage in pulmonary fibrosis

doi: 10.1101/2024.05.27.594700

Figure Lengend Snippet: a , Representative western blot analysis and quantification (right panel) of total O-GlcNAc levels in control (n = 6) and IPF (n = 5) lung lysates revealed significant increase of O-GlcNAc in IPF lungs (violin plot, ** p < 0.01, unpaired t -test). b , c , RT-PCR analysis of MMP10 ( b ) and FN1 ( c ) in OGT-deleted airway epithelial cells treated with IPF-stimuli shows that OGT is required for profibrotic genes expression (n = 6, mean + s.e.m, * p < 0.05, **** p < 0.0001, ANOVA/Tukey’s). d, RT-PCR analysis of COL1A1 in IPF AOs treated with IPF-stimuli and OSMI-4 for 7 days shows decrease of gene expression upon OGT inhibition (n = 7, mean + s.e.m, ** p < 0.01, **** p < 0.0001, ANOVA/Tukey’s). e , ELISA analysis shows a decline in MMP7 secretion in an OGT-dependent manner upon stimulation with IPF-stimuli (n = 8, mean + s.e.m, * p < 0.05, ** p < 0.01, ANOVA/Friedman). f , RT-PCR analysis shows that OGT inhibition attenuates chronically injured epithelial-fibroblast coculture induced COL1A1 expression levels (n = 5, mean + s.e.m, ** p < 0.01, *** p < 0.001, ANOVA/Tukey’s). g , ELISA analysis reveals that OGT inhibition in chronically injured epithelial-fibroblast coculture ameliorates MMP7 secretion (n = 5, mean + s.e.m, * p < 0.05, ANOVA/Tukey’s). h, Quantification of ciliated cell area upon OSMI-4 treatment and chronic injury in epithelial-mesenchymal coculture increased area covered by ciliated cells (n = 3, mean + s.e.m, * p < 0.05, ** p < 0.01, ANOVA/Tukey’s). i , Quantification of average organoid size shows decreased organoid growth upon inhibition of OGT after 10 days (n = 5, mean + s.e.m, ** p < 0.01, ANOVA/Tukey’s). j , Representative immunofluorescence staining of IPF and control AOs treated with IPF-stimuli and OSMI-4 for 7 days shows decrease in aberrant basal cell signature upon OGT inhibition (scale bar 50 µm).

Article Snippet: Measurement of human MMP7 (Biotechne, #DY907) was performed according to manufacturer’s instructions.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Inhibition, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: JUNB O-GlcNAcylation-mediated promoter accessibility of metabolic genes modulates distinct epithelial lineage in pulmonary fibrosis

doi: 10.1101/2024.05.27.594700

Figure Lengend Snippet: a , Representative western blot analysis of the O-GlcNAc immunoprecipitated fraction shows increased levels of O-GlcNAc mark on JUNB in injured airway epithelial cells (n = 3 independent donors). IgG antibody was employed as negative control. b , c , RT-PCR analysis shows diminished expression of pro-fibrotic genes ( COL1A1 ( b ) and MMP10 ( c )) upon transfection of JUNB-LOF (loss-of-function) and fibrotic induction IPF-stimuli in airway epithelial cells (n = 5, mean + s.e.m, * p < 0.05, ** p < 0.01, **** p < 0.0001, ANOVA/Tukey’s) Plasmid was generated by site-specific mutations in the O-GlcNAc sites of JUNB. d , Transduction of JUNB-LOF plasmid in n = 6 IPF AOs led to a decrease in the secretion of MMP7 after 10 days (mean + s.e.m, * p < 0.05, Wilcoxon). e , f , Representative pictures ( e ) and quantification ( f ) of average organoids size of n = 6 IPF AOs was reduced after JUNB-LOF transduction in AOs after 10 days, whereas overexpression of JUNB led to a significant increase in growth (mean + s.e.m, * p < 0.05, ANOVA/Tukey’s).

Article Snippet: Measurement of human MMP7 (Biotechne, #DY907) was performed according to manufacturer’s instructions.

Techniques: Western Blot, Immunoprecipitation, Negative Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation, Generated, Transduction, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mesothelin promotes brain metastasis of non-small cell lung cancer by activating MET

doi: 10.1186/s13046-024-03015-w

Figure Lengend Snippet: MSLN expression is increased in NSCLC patients with BM. A Schematic illustration of the selection process of brain metastasis(BM) derivatives in mice. Parent cells PC9 and H2030 were inoculated into the left ventricle of nude mice to isolate and collect tumor cells with BM. The selection process was carried out twice, and the high-brain metastatic subpopulation (PC9-BrM and H2030-BrM cell lines) were collected. B Differential protein volcano map between PC9-BrM cells and PC9 cells. C Western blot analysis showed that PC9-BrM and H2030-BrM cells with high metastatic activity had higher MSLN protein levels. D Representative images and quantification analysis of MSLN staining in primary lung tumor (LT, n =36) and NSCLC-derived brain metastases (BM, n =34) surgical specimens. (scale bar, 200 μm) . E ELISA detection of MSLN expression in serum of all patients and control groups. HC, healthy controls ( n =24). ELC, early-stage NSCLC ( n =22). BoM, lung cancer with bone metastasis ( n =23). LM, lung cancer with live metastasis ( n =20). LCBM, lung cancer with brain metastasis ( n =42). PBT, primary brain tumor ( n =23). F Kaplan-Meier analysis of the overall survival of 478 lung cancer patients in the GEPIA database. (Data are presented as mean ± SD)

Article Snippet: Target protein concentrations were measured using a Human MMP7 ELISA Kit (Elabscience) and a Human MSLN ELISA Kit (Omnimabs).

Techniques: Expressing, Selection, Western Blot, Activity Assay, Staining, Derivative Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mesothelin promotes brain metastasis of non-small cell lung cancer by activating MET

doi: 10.1186/s13046-024-03015-w

Figure Lengend Snippet: MSLN promotes the migration and invasion of NSCLC cells in vitro . A Representative images and quantitative results of western blotting showing MSLN expression after transfection of PC9-BrM and PC9 cells with lentivirus. B The effect of MSLN expression on the migration capacity in PC9-BrM and PC9 cells assessed in a wound-healing assay (scale bar, 100 µm). C Transwell migration and invasion assays to determine the effect of altered MSLN expression on the migration and invasion of NSCLC cells (scale bar, 200 µm). D, E Western blot analysis of E-cadherin, N-cadherin, Slug and MMP7 expression in PC9-BrM cells and PC9 cells after alteration of MSLN expression. F-G The expression of MMP7 by PC9 cells and PC9-BrM cells was detected by qRT-PCR (F) and ELISA (G). (PC9-NC, PC9 cells transfected with negative control plasmid. PC9-OE, PC9 cells transfected with MSLN plasmid. PC9-BrM-NC, PC9-BrM cells transfected with negative control shRNA. PC9-BrM-SH1, PC9-BrM cells transfected with MSLN-targeted shRNA1. PC9-BrM-SH2, PC9-BrM cells transfected with MSLN-targeted shRNA2. Data are presented as mean ± SD, ns, no significance)

Article Snippet: Target protein concentrations were measured using a Human MMP7 ELISA Kit (Elabscience) and a Human MSLN ELISA Kit (Omnimabs).

Techniques: Migration, In Vitro, Western Blot, Expressing, Transfection, Wound Healing Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Negative Control, Plasmid Preparation, shRNA