Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: FFPE lung tissue sections from control and COPD donors were stained using immunohistochemistry for ECM and ECM-associated proteins with specific signals being detected with Nova red (red) and counterstained with hematoxylin (blue). Sections were scanned at 40x magnification using a digital slide scanner. Scale bar = 400um. Images are representative of protein detection patterns seen in control donors (n=18), SEO-COPD (n=12), and moderate COPD (n=14). ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; FFPE: formalin fixed paraffin embedded; COL1A1: collagen type I α chain 1; COL6A1: collagen type VI α chain 1; COL6A2: collagen type VI α chain 2; COL14A1: collagen type XIV α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Control, Staining, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Binding Assay

Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: ECM proteins in FFPE tissue sections from controls (n=18), SEO-COPD (n=12), and moderate COPD (n=14) were detected using immunohistochemistry. Parenchyma was isolated and analyzed for percentage positive tissue area and mean intensity of positive pixels of expression for each protein. All 44 donors were available for analysis of COL1A1, COL6A2, FBLN2, LTBP4, LUM, DCN, VCAN, AND ELN, while for COL6A1, COL14A1, and FBLN5 43 donors were available. For each protein, regression coefficients (± 95% CI) were obtained following linear regression and a p value of <0.05 was considered significant. Non-transformed variables are plotted first, followed by log transformed variables. The differences in ECM and ECM-associated proteins in COPD tissue were compared to control in terms of A-C) percentage area and D-F) mean intensity of positively stained pixels. Dark blue-colored bars in light blue colored boxes highlight significant differences. ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; FFPE: formalin fixed paraffin embedded; COL1A1: collagen type I α chain 1; COL6A1: collagen type VI α chain 1; COL6A2: collagen type VI α chain 2; COL14A1: collagen type XIV α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Immunohistochemistry, Isolation, Expressing, Transformation Assay, Control, Staining, Formalin-fixed Paraffin-Embedded, Binding Assay

Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: ECM proteins present in FFPE tissue sections from controls (n=18), SEO-COPD (n=12), and moderate COPD (n=14) were detected using immunohistochemistry. Airway walls were isolated and analyzed for percentage area and mean intensity of expression for each protein. The number of airway walls available for the analysis for each protein were COL1A1 (n= 155), COL6A1 (n=150), COL6A2 (n=149), COL14A1 (n=152), FBLN2 (n=158), FBLN5 (n=173), LTBP4 (n=163), LUM (n=158), DCN (n=156), VCAN (n=165), and ELN (n=158). For each protein, regression coefficients (± 95% CI) were obtained following linear regression and a p value of <0.05 was considered significant. Non-transformed variables are plotted first, followed by log transformed variables. The differences in ECM and ECM-associated proteins in COPD tissue were compared to control in terms of A-C) percentage area and D-F) mean intensity of positively stained pixels. Dark blue-colored bars in light blue colored boxes highlight significant differences. ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; FFPE: formalin fixed paraffin embedded; COL1A1: collagen type I α chain 1; COL6A1: collagen type VI α chain 1; COL6A2: collagen type VI α chain 2; COL14A1: collagen type XIV α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Immunohistochemistry, Isolation, Expressing, Transformation Assay, Control, Staining, Formalin-fixed Paraffin-Embedded, Binding Assay

Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: ECM differences noted in the different analyzes throughout this study have been summarized here. In the staining and ECM signatures, red or blue arrows indicate higher or lower proportional levels or component scores in COPD respectively, while they denote positive or negative associations with FEV1 respectively. ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; COL1A1: type I collagen α chain 1; COL6A1: type VI collagen α chain 1; COL6A2: type VI collagen α chain 2; COL14A1: type XIV collagen α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin; FEV1: forced expiratory volume in 1 second.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Staining, Binding Assay

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Impact of fibulin‐5 deficiency on the skin aging process. (A) Schematic representation of the interfollicular epidermis of mouse tail skin. Slow‐cycling epidermal stem cells (SCs) produce the K10 + interscale lineage (orange), and fast‐cycling epidermal SCs produce the K36 + scale lineage (blue). (B, C) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 2‐month‐old versus 30‐month‐old C57BL/6J mice and quantification (C). The white dashed line represents the epidermal–dermal boundary. Scale bars: 50 μm. (D, E) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 3‐month‐old Fbln5 WT versus KO mice and quantification (E). The white dashed line represents the epidermal–dermal boundary and hair follicles. Scale bars: 50 μm. (F) Images of 12‐month‐old Fbln5 WT and KO mice. (G) The body weights of 12‐month‐old Fbln5 WT and KO mice. (H–K) Hematoxylin and eosin staining of sagittal sections of the skin of 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (I, K). Scale bars: 150 μm. Epidermal thickness was measured in interscale and scale regions. (L–O) Whole‐mount staining of BrdU (green, a proliferation marker) and Hoechst (blue) in 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (M, O). Scale bars: 200 μm. (P–U) Whole‐mount staining of K10 (green, interscale lineage), K36 (red, scale lineage), and Hoechst (blue) in 2‐month‐old versus 30‐month‐old C57BL/6J mice and 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (Q, S, U). Scale bars: 200 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (C, E, G, I, K, M, O, Q, S, U). *, p < 0.05; **, p < 0.01; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Immunostaining, Staining, Marker, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Changes in integrin and extracellular matrix expression due to the loss of fibulin‐5. (A) The heatmap shows changes in integrins and ECM proteins in 12‐month‐old Fbln5 WT and KO epidermal stem cells. Genes with a ≥ 2‐fold change are used for analysis. (B) Schematic representation of the epidermal–dermal junction and its associated proteins. (C–V) Immunostaining and quantification of the indicated proteins: Collagen XVII (C–F; green), integrin β1 (G–J; green), integrin α6 (K–N; red) integrin β3 (O–R; green), nectin‐3 (S–V; green), K5 (S–V; gray), and K36 (S–V; red, scale lineage). The white dashed lines represent the epidermal–dermal boundary. Scale bars: 50 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (D, F, H, J, N, P, R, T, V) or Mann–Whitney U test (L). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. The schematic in panel B is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Immunostaining, Two Tailed Test, MANN-WHITNEY

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Extracellular fibulin‐5 enhances YAP activity and fast‐cycling stem cell‐associated gene expression in human keratinocytes. (A–H) Immunostaining of YAP (A, green), SLC1A3 (C, red), Ki‐67 (E, gray), and ASS1 (G, green) in human keratinocytes and quantification (B, D, F, H). Cells are seeded at 150,000, 50,000, and 25,000 cells per well in 12‐well plates and cultured for 48 h before analysis. Scale bars: 50 μm. (I, J) Immunostaining of YAP in primary human keratinocytes and quantification (J). Cells are seeded at 50,000 cells per well in 12‐well plates and cultured for 24 h and then treated with verteporfin or vehicle control for 8 h. Nuclear YAP (%) was calculated as the proportion of cells with nuclear YAP localization among all Hoechst + nuclei. Scale bars: 50 μm. (K–M) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following 8 h of verteporfin treatment. (N, O) Immunostaining of YAP in primary human keratinocytes and quantification (O). Cells are seeded at 300,000 cells per well on collagen IV–coated plates with or without recombinant human fibulin‐5 and cultured to ~80% confluence. The medium is then replaced, and cells are analyzed 8 h later. Scale bars: 50 μm. (P–R) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following culture on plates coated with collagen IV ± fibulin‐5. All data are presented as the mean ± SD. Each dot represents one independent biological replicate. Statistical significance is assessed using a two‐tailed unpaired t ‐test (K, L, P, Q, R), Welch's t ‐test (J, M, O), or one‐way ANOVA (B, D, F, H). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Activity Assay, Gene Expression, Immunostaining, Cell Culture, Control, Quantitative RT-PCR, Recombinant, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Proposed model of cellular and molecular alterations associated with fibulin‐5 deficiency during skin aging. In young skin, slow‐cycling and fast‐cycling epidermal stem cells (SCs) are spatially compartmentalized and give rise to their respective lineages. During aging, decreased fibulin‐5 expression is associated with altered integrin and extracellular matrix (ECM) protein expression, potentially affecting intracellular signaling through fibulin‐5–integrin interactions. Reduced YAP activity is associated with a decrease in the fast‐cycling epidermal stem cell compartment in aged skin and human keratinocytes. The schematic is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Activity Assay

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: LTBP-2 competes with tropoelastin for binding to fibulin-5 and heparin, and is a negative modulator of elastinogenesis.

doi: 10.1016/j.matbio.2013.10.007

Figure Lengend Snippet: Fig. 5. Confocal co-localisation of LTBP-2 with fibulin-5, fibrillin-1, fibronectin and heparan sulfate proteoglycans in fibroblast matrix. Human skin fibroblast cultures were cultured for 21 days and then stained for confocal microscopy with antibodies to LTBP-2 in combination with antibodies to fibulin-5, fibrillin-1, fibronectin, perlecan or syndecan-4. Primary antibody was detected using an appropriate secondary antibody conjugated to fluorophore Alexa488 (red) or Cy3 (green). (see Experimental section). A,D,G,J, and M, rabbit anti-[LTBP-2 peptide] antibody 3504 [16 μg/ml]; B, mouse anti-fibrillin-1 antibody MAB1919 [5 μg/ml]; C = A and B merged; E, mouse anti-fibulin-5 antibody MAB3095 [25 μg/ml]; F = D and E merged; H, mouse anti-[cellular fibronectin] antibody ab6328 [10 μg/ml]; I = G and H merged; K, mouse anti-perlecan antibody [2.5 mg/ml]; L = J and K merged; N, mouse syndecan-4 antibody [4 μg/ml]; O = M and N merged; P, rabbit IgG [15.6 μg/ml] control for A,D,G,J and M; Q, mouse IgG (25 μg/ml) control for B,E,H,K and N; R = P and Q merged. Bar = 200 μM.

Article Snippet: Anti-fibulin5 (DANCE) antibody MAB3095 was purchased from R and D systems Inc., Minneapolis, MN, USA.

Techniques: Cell Culture, Staining, Confocal Microscopy, Control

Journal: Cancers

Article Title: Clinical and Biological Significances of FBLN5 in Gastric Cancer.

doi: 10.3390/cancers15020553

Figure Lengend Snippet: Figure 1. The associations between the FBLN5 mRNA expression levels and prognostic and clinico- pathological features. (A) The PCA analysis assessed the grouping situation of patients with different levels of FBLN5 expression. (B) Overall survival analysis of patients with high- and low-expression levels of FBLN5 in the HMU-TCGA database (p < 0.001). (C) Overall survival analysis of patients with high- and low-expression levels of FBLN5 in the GEO database (p < 0.001). (D) DSS analysis of patients with high- and low-expression groups of FBLN5 (p < 0.05). (E) PFI analysis of patients with high- and low-expression groups of FBLN5 (p < 0.05). (F) Analysis of the differential expression of FBLN5 in GC (619 cases) and normal tissues (300 cases). (G–I) The expression of FBLN5 in GC tissues in patients with different grades of disease (p < 0.05). *** p < 0.001. (HMU, Harbin Medical University; TCGA, the Cancer Genome Atlas; GEO, gene expression omnibus; GC, gastric cancer; DSS, disease-specific survival; PFI, progress-free interval).

Article Snippet: Next, the FBLN5 rabbit polyclonal antibody (ABclonal, Woburn, MA, USA) was diluted at a ratio of 1:150, dropped on the glass slide, and incubated overnight at 4 ◦C (not more than 16 h).

Techniques: Expressing, Quantitative Proteomics, Gene Expression

Journal: Cancers

Article Title: Clinical and Biological Significances of FBLN5 in Gastric Cancer.

doi: 10.3390/cancers15020553

Figure Lengend Snippet: Figure 2. Analyses of biological functions of FBLN5. (A) Analysis of the differential genes between groups expressing high and low levels of FBLN5 using the limma package. Representative GO enrichment analysis between groups expressing high and low levels of FBLN5 based on TCGA- GC data, which included the following: (B) Biological processes (BP) associated with FBLN5 gene functions, (C) Cellular components (CC) of FBLN5 gene functions, (D) Molecular functions (MF) associated with FBLN5 gene functions. (E) Representative KEGG enrichment analysis between groups expressing high and low levels of FBLN5 based on TCGA-GC data. (F) GSEA analysis of the group expressing high levels of FBLN5 using hypoxia, angiogenesis, TGF-β, EMT, and apoptosis signals (ALL|normalized enrichment score (NES)| > 1, nominal (NOM) p-value < 0.05 and FDR q-value < 0.25). (G) PPI analysis of FBLN5-associated proteins.

Article Snippet: Next, the FBLN5 rabbit polyclonal antibody (ABclonal, Woburn, MA, USA) was diluted at a ratio of 1:150, dropped on the glass slide, and incubated overnight at 4 ◦C (not more than 16 h).

Techniques: Expressing

Journal: Cancers

Article Title: Clinical and Biological Significances of FBLN5 in Gastric Cancer.

doi: 10.3390/cancers15020553

Figure Lengend Snippet: Figure 3. Predicting the prognosis of GC patients based on the mRNA expression levels of FBLN5. (A–G) ROCs analyzed the prognostic ability of FBLN5, ELN, LOXL1, LOXL2, LOXL3, and LOXL4 mRNA expression levels. (H) Prognostic risk score for FBLN5 expression, ELN expression, and LOX expression. (I) Assessment of the patient’s OS based on a prognostic risk score. (J) Risk factor graph to visualize the trend of prognostic model. (K) Nomogram prediction model based on risk score, pTNM stage, and age. (L) ROC analysis based on FBLN5 expression, risk score, and nomograph prediction model. (OS: overall survival).

Article Snippet: Next, the FBLN5 rabbit polyclonal antibody (ABclonal, Woburn, MA, USA) was diluted at a ratio of 1:150, dropped on the glass slide, and incubated overnight at 4 ◦C (not more than 16 h).

Techniques: Expressing

Journal: Cancers

Article Title: Clinical and Biological Significances of FBLN5 in Gastric Cancer.

doi: 10.3390/cancers15020553

Figure Lengend Snippet: Figure 4. Relationships between FBLN5 and immunity. (A) CIBERSORT and (B) TIMER algorithms preliminarily predicted the relationship between the FBLN5 expression levels and immune infiltration in each tumor sample. The (C) ESTIMATE algorithm estimated the content of immune and stromal cells in GC, and helped to predict the (D) immune score, (E) stromal score, and (F) tumor purity. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Next, the FBLN5 rabbit polyclonal antibody (ABclonal, Woburn, MA, USA) was diluted at a ratio of 1:150, dropped on the glass slide, and incubated overnight at 4 ◦C (not more than 16 h).

Techniques: Expressing

Journal: Cancers

Article Title: Clinical and Biological Significances of FBLN5 in Gastric Cancer.

doi: 10.3390/cancers15020553

Figure Lengend Snippet: Figure 5. Relationships between FBLN5 and tumor progression. (A) Chemotherapy analysis based on FBLN5 expression levels. (B) EMT score. (C) Mutations in FBLN5 during the regulation of cancer progression. *** p < 0.001.

Article Snippet: Next, the FBLN5 rabbit polyclonal antibody (ABclonal, Woburn, MA, USA) was diluted at a ratio of 1:150, dropped on the glass slide, and incubated overnight at 4 ◦C (not more than 16 h).

Techniques: Expressing

Journal: Cancers

Article Title: Clinical and Biological Significances of FBLN5 in Gastric Cancer.

doi: 10.3390/cancers15020553

Figure Lengend Snippet: Figure 6. FBLN5 expression levels and prognostic model establishment. (A) FBLN5 immunohisto- chemistry and H&E staining of GC TMA, ×50 and ×400 total magnification. (A1) Well-differentiated

Article Snippet: Next, the FBLN5 rabbit polyclonal antibody (ABclonal, Woburn, MA, USA) was diluted at a ratio of 1:150, dropped on the glass slide, and incubated overnight at 4 ◦C (not more than 16 h).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Cancers

Article Title: Clinical and Biological Significances of FBLN5 in Gastric Cancer.

doi: 10.3390/cancers15020553

Figure Lengend Snippet: Figure 7. Application of FBLN5 in other cancers. (A) Expression levels of FBLN5 in various cancers. (B) Prognostic analysis of FBLN5 in various cancers. (C) TIMER algorithm and (D) CIBERSORT algorithm preliminarily predicted the relationship between the expression level of FBLN5 and immune infiltration in various types of tumors. (E) Pan-cancer analysis of FBLN5-related immune checkpoints, along with (F) tumor purity, (G) TMB, and (H) MSI analyses to evaluate the prospect of using FBLN5 for targeted pan-cancer treatment. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Next, the FBLN5 rabbit polyclonal antibody (ABclonal, Woburn, MA, USA) was diluted at a ratio of 1:150, dropped on the glass slide, and incubated overnight at 4 ◦C (not more than 16 h).

Techniques: Expressing