Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: CCN1 secretion and cleavage regulate the lung epithelial cell functions after cigarette smoke

doi: 10.1152/ajplung.00102.2014

Figure Lengend Snippet: CSE-induced CCN1 augments matrix metalloproteinase (MMP)-1 secretion. A: effects of CCN1 knockdown on MMP1. Beas2B cells were treated with control or CCN1 siRNA, followed with 10% CSE. MMP1 levels in supernatants were determined using ELISA 4 h later. B: the soluble factor and extracellular vehicles (EVs) were isolated from CSE-stimulated Beas2B cells. Beas2B cells were treated with control or CCN1 siRNA. The Beas2B cells were stimulated by the soluble factor (10 μg) and EV (10 μg). MMP1 was then determined using ELISA. C: Beas2B cells were treated with recombinant CCN1 (1 μg/ml), plasmin (0.1 μg/ml), and CCN1 + plasmin. After 8 h, MMP1 was determined using ELISA. CCN1 and plasmin were preincubated 1 h at 37°C before adding to the cells. D: Beas2B cells were pretreated for 1 h with TLCK before CSE, and MMP1 was determined using ELISA 8 h later. E: level of MMP1 after treatment of NH2-terminal or COOH-terminal CCN1 fragments (each 1 μg) in Beas2B cells. Supernatant (Sup) was taken after 24 h and measured MMP1. F: cell survival after treatment of NH2-terminal or COOH-terminal CCN1 fragments for 48 h in Beas2B cells. Cell survival was measured by CellTiter-Glo Luminescent cell viability assay. *P < 0.05 compared with control for A and B, sham for C and F, and CSE for D and E. **P < 0.05 compared with CCN1 and plasmin for C and NH2-terminal for E. Each panel represents at least 3 repeats with similar results.

Article Snippet: The human IL-8 ELISA kit was purchased from Thermo (Rockford, IL), and human VEGF and MMP-1 duoset was from R&D Systems and followed the manufacturer's instructions. .

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Recombinant, Cell Viability 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, 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: Frontiers in Physiology

Article Title: Classical and Non-classical Fibrosis Phenotypes Are Revealed by Lung and Cardiac Like Microvascular Tissues On-Chip

doi: 10.3389/fphys.2021.735915

Figure Lengend Snippet: TGF-β treatment induces differential stromal-cell matrix remodeling. (A) Schematic diagram of TGF-β treatment regimens and subsequent nanoindentation experiments. (B) Measured effective Young’s modulus of the microvessels for the low concentration/short-term and high concentration/long-term conditions. (C) Relative αSMA expression in the different treatment regimens determined by Western Blot (for iPSC-EC low conc./short-term n = 3 samples were pooled). (D) MMP 1 and MMP 9 expressed in TGF-β treated microvascular tissues measured by ELISA. Dashed region is the mean value for media. Significance is shown by * P < 0.05, using a one-way ANOVA and a subsequent Tukey means comparison test. Box plots demonstrate SD (outer whiskers) and SE (box edge).

Article Snippet: Using two separate DuoSet ELISA KITs (R&D systems, DY901B and DY911), the concentration of MMP-1 and MMP-9 were determined for TGF-β treated conditions per the manufacturer’s instructions.

Techniques: Concentration Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison