Journal: PLoS ONE

Article Title: C/EBPβ-Thr217 Phosphorylation Signaling Contributes to the Development of Lung Injury and Fibrosis in Mice

doi: 10.1371/journal.pone.0025497

Figure Lengend Snippet: A. Immunoblots for RSK, C/EBPβ-phospho-Thr217, procaspase 8 and C/EBPβ were performed on C/EBPβ immunoprecipitates from activated primary human LMF lysates as described in . RSK and phosphorylated C/EBPβ were induced in activated LMF but decreased in activated LMF treated with the ERK1/2 inhibitor (10 µg for 24 hr) or with the C/EBPβ peptide (200 µg for 24 hr). Inactive procaspase 8 was associated with phosphorylated C/EBPβ in untreated, activated LMF, while active caspase 8 was associated with unphosphorylated C/EBPβ in activated LMF treated with the ERK1/2 inhibitor or with the C/EBPβ peptide. Human LMF expressed full-length C/EBPβ from the second AUG . β-Actin was used to correct for lung lysate input. We performed single analysis of the samples. Results from triplicate samples of two independent experiments are shown. B. The ERK1/2 inhibitor decreased the fold association of RSK with C/EBPβ (n: 6 per group; 0.07+/−0.01, P <0.0001), and the fold expression of C/EBPβ (n: 6 per group; 0.05+/−0.006, P <0.0001) and C/EBPβ-PhosphoThr266 (n: 6 per group; 0.33+/−0.078, P <0.0001) in activated, human lung fibroblasts. There was also an increased fold association between unphosphorylated human C/EBPβ and active caspase 8 (n: 6 per group; active caspase 8; 5.70+/−0.59, P <0.0001). The cell permeant Ac-KAla217VD-CHO peptide also inhibited C/EBPβ expression (n: 6 per group; 0.12+/−0.01, P <0.0001), the phosphorylation of C/EBPβ-Thr266 (n: 6 per group; 0.17+/−0.03, P <0.0001), the association of RSK with C/EBPβ (n: 6 per group; 0.24+/−0.08, P <0.0001) and increased the association of RSK with C/EBPβ (n: 6 per group; 0.24+/−0.08, P <0.0001). We performed single analysis of the samples. C. α-SMA and TGF-β were induced in activated LMF but the expression of these fibrogenic genes was inhibited by treatment with the ERK1/2 inhibitor (10 µg for 24 hr) or with the peptide (200 µg for 24 hr). β-Actin was used to correct for lung lysate input. Representative results from two independent studies. D. The ERK1/2 inhibitor decreased the fold expression of α-SMA (n: 6 per group; 0.24+/−0.11, P <0.0001) and TGF-β1 (n: 6 per group; 0.14+/−0.02, P <0.0001). The cell permeant Ac-KAla217VD-CHO peptide also inhibited the fold expression of α-SMA (n: 6 per group; 0.24+/−0.11, P <0.0001) and TGF-β1 (n: 6 per group; 0.14+/−0.02, P <0.0001). We performed single analysis of the samples. E. Annexin-V-PE binding in vivo in activated LMF was increased after treatment with the ERK1/2 inhibitor (20 µg for 8 hr) or with the peptide (200 µg for 24 hr). Values are the percentage of cells expressing annexin-V-PE binding as described in . Activated, human lung fibroblasts treated with the ERK1/2 inhibitor (n: 6; 66.33+/−5.68%, P <0.0001) or with the Ac-KAla217VD-CHO peptide (n: 6; 61.00+/−9.27%, P <0.0001) displayed increased percent annexin-V binding compared to control (n: 6; 4.15+/−0.94%). We performed single analysis of the samples. Results from triplicate samples of three independent experiments are shown.

Article Snippet: Primary human lung fibroblasts isolated from normal human lung parenchyma (Cell Applications; San Diego, CA) were cryopreserved at first passage and can be cultured and propagated at least 12 population doublings.

Techniques: Western Blot, Expressing, Phospho-proteomics, Binding Assay, In Vivo, Control

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: TAOB-CMs increases lung cancer migration and EMT. TAOB-CMs enhanced the migratory ability of A549 and CL1–0 lung cancer cells. A and B, TAOB-CMs enhanced cell migratory ability, as determined by scratch wound healing assay (A) and Transwell system (B). C, TAOB-CMs enhanced cell invasion ability. D, TAOB-CMs caused EMT in cancer cells. E, the expression of BMP-2 in OB-CM, For A, the migration ability of lung cancer cells was assessed by wound healing assay. OB-CM (control group) and TAOB-CMs (20%) act as a chemoattractant of cancer migration. Quantification of cell migration was carried out by measuring the distance between the migratory fronts of cells in four random selected microscopic fields for each condition and time point. The degree of cell movement is expressed as the percentage of wound closure as compared with the zero time point. For B and C, the invasiveness and migration ability of A549 and CL1–0 cells were quantified by QCMTM 24-well cell migration and invasion assay. The cells were seeded in the upper inset, and the OB-CM (control group) and TAOB-CMs (20%) acted as the chemoattractant for cancer migration and invasion. For D, A549 and CL1–0 cells were treated with TAOB-CMs (20%) for 24 h, and then the expression of various proteins was assessed by immunoblot assay. For E, primary osteoblasts were treated RPMI 1640 (20%), A549-CM (20%), and CL1–0-CM for 24 h. The BMP-2 levels were assessed by BMP-2 ELISA kits. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Migration, Wound Healing Assay, Expressing, Invasion Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: BMP-2 is involved in TAOB-CM-mediated enhancement of migration and EMT in lung cancer. A and B, BMP-2 increased migratory ability, as determined by scratch wound healing assay (A) and Transwell system (B). C and D, BMP-2 increased the invasion ability (C) and EMT (D) of A549 and CL1–0 cells. E and F, noggin decreased TAOB-CM-mediated cell migration (E) and EMT (F). The migration ability of lung cancer cells was assessed by wound healing assay, in accord with the description under “Experimental Procedures.” BMP-2 (20 ng/ml for EMT assay) acts as the chemoattractant for cancer migration. For E and F, A549 and CL1–0 cells were pretreated with or without noggin for 1 h, and then OB-CM and TAOB-CMs were added for another 24 h. Cell migration was assessed by wound healing assay, and the expression of various proteins was determined by immunoblot assay. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Migration, Wound Healing Assay, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: Sera from lung cancer patients increase lung cancer migration. A, the levels of BMP-2 in lung cancer patient sera. B, lung cancer sera enhance the migratory ability of lung cancer cells. C, depletion of BMP-2 decreased lung cancer patient serum-mediated cell migration. The levels of BMP-2 were assessed by ELISA. Horizontal bars represent means. The cells were treated with or without noggin for 1 h, and then culture medium containing healthy donor sera (15%) or lung cancer patient sera (15%) was added for another 24 h. Cell migration was assessed by wound healing assay. For C, BMP-2 depleted from lung cancer patient serum was performed using anti-BMP-2 and antibodies (4 μg/ml) and Sepharose A/G beads, following regular immunoprecipitation techniques. The migration ability of A549 and CL1–0 cells were quantified by QCMTM 24-well cell migration assay kit. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Immunoprecipitation, Cell Migration Assay

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: TAOB-CMs and BMP-2 increase the activation of MAPK and elevate the expression of Runx2 and Snail. A and B, TAOB-CMs (A) and BMP-2 (B) increase the phosphorylation of SMAD, ERK, and p38. C and D, TAOB-CMs (C) and BMP-2 (D) enhance the expression of Runx2 and Snail protein. Cells were treated with OB-CM (20%), TAOB-CMs (20%), or BMP-2 (20 ng/ml) for the indicated times. The expressions of various proteins were determined by immunoblot assay. E, TAOB-CMs and BMP-2 enhance the expression of Runx2 and Snail mRNA. The cells were treated with OB-CM (20%), TAOB-CMs (20%), or BMP-2 (20 ng/ml) for a specific time (3 h for snail and 12 h for E-cadherin). The expressions of mRNA were determined by quantitative PCR. F, noggin decreases TAOB-CM-mediated MAPK activation and Runx2 and Snail up-regulation. The cells were treated with OB-CM (20%), TAOB-CMs (20%), or BMP-2 (20 ng/ml) for the indicated times. The expressions of mRNA and various proteins were determined by quantitative PCR and immunoblot assay. For F, A549 and CL1–0 cells were pretreated with or without noggin for 1 h and then treated with BMP-2 (20 ng/ml) for 6 h. The expression of various proteins was then assessed by immunoblot assay. The data shown are representative of three independent experiments. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Activation Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: Runx2 is the upstream regulatory factor of Snail. A and B, inhibition of Runx2 decreases BMP-2-mediated Snail up-regulation and E-cadherin down-regulation (A), as well as cell migration (B). Cells were transfected with pLKO-AS2 or pLKO-AS2-RUNX2 shRNA. Stable clones were created by puromycin selection, and the efficacy of shRNA was assessed by RT-PCR. Cells were treated with BMP-2 (20 ng/ml) for the specified times (cell migration, 24 h; Runx2 and Snail, 6 h; E-cadherin, 24 h). Then the expression of various proteins was then assessed by immunoblot assay. C, overexpression of Snail reversed the inhibitory effect of Runx2 shRNA on BMP-2-mediated cell migration. Runx2-transfected A549 and CL1–0 cells were transected with pCMV or pSnail plasmid, and stable clones were established by G418 and puromycin. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05). The data shown are representative of three independent experiments.

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Inhibition, Migration, Transfection, shRNA, Clone Assay, Selection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Over Expression, Plasmid Preparation

Journal: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: Exhaled breath condensates from healthy children induce cell death of in vitro cultured cells by activation of apoptosis

doi: 10.5114/ada.2019.87087

Figure Lengend Snippet: A – The culture of human lung fibroblasts (HLF) in the phase contrast microscope after 4-hour incubation with double distilled water (DDW), used as a test control (upper picture) or with a representative EBC sample (bottom picture). B – The membranes of Human Apoptosis Array exposed to HLF lysates incubated for 4 h with DDW (left picture) or a representative EBC sample (right picture). Each pair of spots in white frames represents a respective target molecule. The numbers at the frames correspond to respective molecules on graph C: 1 – CD95/Fas, 2 – pro-caspase-3, 3 – cleaved caspase-3, 4 – SMAC/Diablo, 5 – Hsp27, 6 – Hsp70. The brightest spots correspond to reference markers (dashed line frames). C – Relative expressions of selected apoptosis pathway-related proteins with the highest signal density, calculated as the percent of reference spots

Article Snippet: The normal human lung fibroblasts (HLF), purchased from Cell Applications Inc. (San Diego, CA), passages 4 th to 8 th , and murine endothelial cell line C166 (ATCC ® CRL-2581TM) from American Type Culture Collection (ATCC, Manassas, VA), were used for in vitro studies.

Techniques: Microscopy, Incubation, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Top differentially expressed genes in flow conditioned primary human lung microvascular endothelial cells (HLMVEC) following exposure to heparinase III (HepIII) relative to vehicle control. Messenger RNA was collected from confluent monolayers of HLMVEC that were conditioned with 15 dyn/cm 2 for 48 h followed by exposure to heparinase III 500 mU/mL or vehicle for 6 h while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). (A) Heatmap representing top 40 differentially expressed genes in HLMVEC between heparinase III and vehicle. Each treatment group contains n = 2 RNA samples that were combined from HLMVEC within two ibidi channel slides, thus representing a total of n = 4 per condition. Colors represent gene expression z-score with red corresponding to upregulated and blue to downregulated. (B) Volcano plot depicting differential gene expression between HLMVEC exposed to heparinase III (positive log2 fold change) and vehicle (negative log2 fold change). Red genes meet figure thresholds of p ≤ 1 × 10 −3 and log2 fold change ≥|1| for the purposes of visualization. (C) Expression of the flow-responsive genes Krüppel-like factor 2 and 4 ( KLF2 , 4 ), endothelial nitric oxide synthase ( NOS3 ) and solute carrier family nine isoform A3 regulatory factor 2 ( SLC9A3R2 ) is reduced following heparinase III treatment. (D) Expression of angiopoietin-2 ( ANGPT2 ), endothelial cell-specific molecule-1 ( ESM1 , also known as endocan), and thrombospondin ( THBS1 ), markers of endothelial cell activation, is increased following heparinase III treatment.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Control, Shear, Gene Expression, Expressing, Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Targeted representation of gene set enrichment analysis (GSEA) in flow conditioned (15 dyn/cm 2 for 48 h) primary human lung microvascular endothelial cells (HLMVEC) after 6-h exposure to vehicle or heparinase III (HepIII, 500 mU/mL) while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). GSEA was performed using (A) GO: Biological Process and (B) KEGG datasets. Figure displays up to twenty pathways from GSEA that are most relevant to endothelial cell organization and function with lowest False discovery rate (FDR)-adjusted p values (FDR q value). Pathways were organized according to their contribution to cellular maintenance and bioenergetics; cell organization and adhesion; angiogenesis and wound healing; or response to biophysical cues. Pathways on presented on the left were enriched in HLMVEC after exposure to vehicle whereas pathways on the right were enriched in HLMVEC after exposure to heparinase III. Circle size corresponds with number of genes present in experimental samples that overlap with respective dataset pathways, and circle shading represents the −log10 (FDR q value) with darker shades representing lower q values.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Shear

Journal: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Differentially expressed genes that govern synthesis of heparan sulfate proteoglycans and glycosaminoglycans in flow conditioned (15 dyn/cm 2 for 48 h) primary human lung microvascular endothelial cells treated for 6 h with vehicle or heparinase III (HepIII, 500 mU/mL) while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). (A) Of the heparan sulfate proteoglycans found in the vascular endothelial apical glycocalyx, expression of syndecan 3 ( SDC3 ) and SDC4 were downregulated by heparinase III treatment. (B) Of the enzymes regulating hyaluronan expression in the endothelial glycocalyx, hyaluronan synthase isoform 2 ( HAS2 ) was upregulated while hyaluronidases 1 and 2 ( HYAL1 , 2 ) were downregulated by heparinase III treatment. (C) Of the enzymes that synthesize chondroitin sulfate expressed in the endothelial glycocalyx (commonly observed in SDC1 and SDC3) and that modify its sulfation, chondroitin sulfate synthase isoform 3 ( CHSY3 ) and chondroitin sulfate N -acetylgalactosaminylsulfotransferase isoform 1 ( CSGALNACT1 ) were upregulated while carbohydrate sulfotransferase isoform 15 ( CHST15 , catalyzing 6- O -sulfation of 4- O -sulfated N -acetylgalactosamine in chondroitin sulfate disaccharides) was downregulated following heparinase III treatment. (D) Of the enzymes that synthesize and modify heparan sulfate expressed in the endothelial glycocalyx, expression of N -deacetylase/ N -sulfotransferase isoform 1 ( NDST1 ) and glucuronic acid C5-epimerase ( GLCE ) (which also contributes to glucuronic acid epimerization to iduronic acid in chondroitin sulfate) were downregulated while heparan sulfate 3- O -sulfotransferase isoform 1 ( HS3ST1 ) and heparan sulfate 6- O -sulfotransferase isoform 3 ( HS6ST3 ) were upregulated following heparinase III treatment. We also found that heparinase III treatment suppressed heparanase ( HPSE ) expression. False discovery rate (FDR)-adjusted p values (FDR q values) are presented.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Shear, Expressing, Histone Deacetylase Assay