Journal: Communications biology

Article Title: Macrophage-derived GPNMB trapped by fibrotic extracellular matrix promotes pulmonary fibrosis.

doi: 10.1038/s42003-022-04333-5

Figure Lengend Snippet: Fig. 3 Increased GPNMB protein level was involved in the effect of fibrotic ECM on lung fibroblast activation. a The effect of the GPNMB protein (0.1 μg/mL) on normal fibroblast viability; n ≥5 independent experiments, ***p < 0.001, **p < 0.01. b The effect of GPNMB (0.1 μg/mL) on fibroblast proliferation; n ≥5 independent experiments, *p < 0.05. c, d Immunofluorescence staining was used to evaluate the effect of GPNMB (0.1 μg/mL) on fibroblast proliferation; scale bar = 200 μm, n ≥5 independent experiments, **p < 0.01. e, f The effect of GPNMB (0.1 μg/mL) on fibroblast migration; scale bar = 100 μm, n ≥5 independent experiments, ***p < 0.001. g The effect of GPNMB (0.1 μg/mL) on fibroblast activation; n ≥5 independent experiments, *p < 0.05. h, i The effect of fibrotic ECM and a GPNMB-neutralizing antibody (0.5 μg/mL) on fibroblast migration; scale bar = 100 μm, n ≥5 independent experiments, ***p < 0.001, *p < 0.05. Nor-ECM, normal ECM; Fib-ECM, fibrotic ECM; Ab, GPNMB-neutralizing antibody.

Article Snippet: Mouse lung fibroblast cells (MLg [Mlg 2908] [ATCC® CCL206TM]) were cultured in DMEM containing fetal bovine serum (FBS, 10%) and penicillin–streptomycin (PS).

Techniques: Activation Assay, Staining, Migration

Journal: Communications biology

Article Title: Macrophage-derived GPNMB trapped by fibrotic extracellular matrix promotes pulmonary fibrosis.

doi: 10.1038/s42003-022-04333-5

Figure Lengend Snippet: Fig. 5 Macrophage deletion via clodronate liposomes relieved PF and fibrotic ECM affected normal fibroblast behaviors in silicosis mice. a Design of the macrophage deletion experiment. b CT images, H&E staining, and Masson’s trichrome staining of lung tissue; scale bar = 200 μm. c H&E staining and Masson trichrome staining of ECM; scale bar = 200 μm. d, e Changes in cell viability and fibroblast numbers in the fibrotic ECM of silicosis mice after macrophage deletion; n ≥5 independent experiments, ***p < 0.001, **p < 0.01. f, g The effect of macrophage deletion on fibroblast migration induced by fibrotic ECM; scale bar = 200 μm, n ≥5 independent experiments, **p < 0.01. NS, normal saline.

Article Snippet: Mouse lung fibroblast cells (MLg [Mlg 2908] [ATCC® CCL206TM]) were cultured in DMEM containing fetal bovine serum (FBS, 10%) and penicillin–streptomycin (PS).

Techniques: Liposomes, Staining, Migration, Saline

Journal: Communications biology

Article Title: Macrophage-derived GPNMB trapped by fibrotic extracellular matrix promotes pulmonary fibrosis.

doi: 10.1038/s42003-022-04333-5

Figure Lengend Snippet: Fig. 7 The CD44/Serpinb2 pathway is related to changes in fibroblast function driven by fibrotic ECM. a Heatmap of differentially expressed mRNAs between fibroblast in different types of ECM. b Volcano plot of differentially expressed proteins between fibroblasts in different types of ECM; |log2(fc)| > 1, p < 0.05. c The KEGG pathways analysis of the upregulated mRNAs. d The GO enrichment analysis of the upregulated mRNAs. e Protein interaction network analysis between GPNMB and Serpinb2. f The change in Serpinb2 protein levels after GPNMB treatment; n ≥5 independent experiments. g The expression level of CD44 gene in different groups on the spatial plots. h The change in CD44 levels in the four groups, as determined by spatial transcriptomics. i CD44 protein levels in fibroblasts after TGFβ1 (5 ng/mL) treatment; n ≥5 independent experiments. NS, normal saline; Nor-ECM, normal ECM; Fib- ECM, fibrotic ECM.

Article Snippet: Mouse lung fibroblast cells (MLg [Mlg 2908] [ATCC® CCL206TM]) were cultured in DMEM containing fetal bovine serum (FBS, 10%) and penicillin–streptomycin (PS).

Techniques: Expressing, Saline

Journal: PLoS ONE

Article Title: Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models

doi: 10.1371/journal.pone.0063121

Figure Lengend Snippet: A. Schematic representation of one well from a 96-well-plate used for the invasion assay and filled with an approximately 300 µm thick 3D collagen matrix. MLg fibroblasts were seeded on top of the collagen matrix and left for invading the matrix at 37°C for 72 h. B. Maximum intensity projection (x-z) from a z-stack taken with a confocal laser scanning microscope from DAPI stained MLg fibroblasts that either invaded the 3D collagen matrix or stayed on top (top panel). The bottom panel shows a spot analysis done with the Imaris (Bitplane) software of the z-stack shown in the upper panel. Invaded fibroblasts (above the red line) are depicted as yellow and non-invaded ones (below the red line) as grey spheres. Scale bar, 50 µm. C. Spot analysis of an invasion assay using A549 epithelial cells as non-invading cells (top panel, grey spheres) and MLg fibroblasts either untreated (middle panel) or treated with 50 ng/ml EGF (bottom panel). Invasion depth is color coded from yellow (non-invaded) to red (invaded). D. Quantitation and statistical evaluation of the amount of MLg fibroblasts that invaded the 3D collagen gel. Data shown represent mean values (± s.d.) from at least three independent experiments (A549: n = 3, CCL206: n = 5). **p<0.01 and ***p<0.001. E. Quantitation and statistical evaluation of the invasion depth of MLg fibroblasts. Data shown represent mean values (± s.d.) from three independent experiments (n = 3). *p<0.05, **p<0.01 and ns = not significant. F. Quantitation and statistical evaluation of the amount of primary human fibroblasts (pHF) that invaded the 3D collagen gel. Data shown represent mean values (± s.d.) from fibroblasts isolated from biopsies/resections of COPD patients (n = 6). **p<0.01.

Article Snippet: Mouse lung fibroblasts MLg (Mlg 2908) were purchased from ATCC (CCL-206) and cultivated in DMEM/HAM’s F12 (catalog# E15-813, PAA) medium containing 10% FBS.

Techniques: Invasion Assay, Laser-Scanning Microscopy, Staining, Software, Quantitation Assay, Isolation

Journal: PLoS ONE

Article Title: Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models

doi: 10.1371/journal.pone.0063121

Figure Lengend Snippet: Maximum intensity projection image of two time points (10 hours and 2 days 14 hours, top and bottom panel, respectively) taken from a confocal 4D time-lapse movie. Fibroblasts were stained with the cell tracker dye CMTPX. Arrows in the bottom panel indicate invaded cells. Scale bar, 50 µm.

Article Snippet: Mouse lung fibroblasts MLg (Mlg 2908) were purchased from ATCC (CCL-206) and cultivated in DMEM/HAM’s F12 (catalog# E15-813, PAA) medium containing 10% FBS.

Techniques: Staining

Journal: PLoS ONE

Article Title: Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models

doi: 10.1371/journal.pone.0063121

Figure Lengend Snippet: Representative confocal images, quantitation and statistical evaluation of cell shapes of MLg fibroblasts found either within (3D) or on top (Top) of the 3D collagen gel compared to fibroblastic cells cultured on conventional 2D plastic surfaces (2D). Data shown represent mean values (± s.d.) from randomly chosen cells (n = 47–73). ***p<0.001 and ns = not significant. Scale bar, 20 µm.

Article Snippet: Mouse lung fibroblasts MLg (Mlg 2908) were purchased from ATCC (CCL-206) and cultivated in DMEM/HAM’s F12 (catalog# E15-813, PAA) medium containing 10% FBS.

Techniques: Quantitation Assay, Cell Culture

Journal: PLoS ONE

Article Title: Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models

doi: 10.1371/journal.pone.0063121

Figure Lengend Snippet: A. Quantitation of the diffusion of FITC, Phalloidin, antibodies IgG-488 and IgG-568 over time. The fluorescent signals were measured by time lapse on a confocal laser scanning microscope. In the diagram the fluorescent signal is depicted as mean grey values (± s.d.) from three independent experiments (n = 3). B. Confocal image of one MLg fibroblast that invaded approximately 100–200 µm into the 3D collagen gel. Cells were stained for DAPI, Phalloidin and an antibody to CD29 (Integrin β1). Boxed areas show a magnified view of the central part of the spindle-shaped cell. The white arrow indicates an elongated fibrillar adhesion-like structure in the CD29 staining that co-localizes with actin stress fibers (Phalloidin). Scale bars, 20 µm.

Article Snippet: Mouse lung fibroblasts MLg (Mlg 2908) were purchased from ATCC (CCL-206) and cultivated in DMEM/HAM’s F12 (catalog# E15-813, PAA) medium containing 10% FBS.

Techniques: Quantitation Assay, Diffusion-based Assay, Laser-Scanning Microscopy, Staining

Journal: PLoS ONE

Article Title: Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models

doi: 10.1371/journal.pone.0063121

Figure Lengend Snippet: A. Scheme of a modified invasion assay using a porous membrane (depicted in blue color) of cell culture inserts to separate invading from non-invading MLg fibroblasts. Fibroblasts were seeded on top of the collagen matrix and left for invading the matrix at 37°C for 72 h. Separation of membrane and collagen matrix is followed by RNA and protein extraction with subsequent qRT-PCR, gene-microarray and protein analysis. B. Differential mRNA expression analysis (qRT-PCR) of MMP13, CXCR4 and FSP-1 in invasive MLg fibroblasts. Data shown represent the fold difference (FD = 2 ΔΔCp ) between invading (inv.) versus non-invading (non-inv.) MLg fibroblasts. Data shown represent mean values (± s.d.) from three independent experiments (n = 3). *p<0.05 and ***p<0.001. C. Representative Western blot of cell lysates comparing MMP13 levels from invading (inv.) versus non-invading (non-inv.) MLg fibroblasts.

Article Snippet: Mouse lung fibroblasts MLg (Mlg 2908) were purchased from ATCC (CCL-206) and cultivated in DMEM/HAM’s F12 (catalog# E15-813, PAA) medium containing 10% FBS.

Techniques: Modification, Invasion Assay, Membrane, Cell Culture, Protein Extraction, Quantitative RT-PCR, Microarray, Expressing, Western Blot