human mmp 7 elisa  (R&D Systems)

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