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: 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: Scientific Reports

Article Title: Plant miRNA osa-miR172d-5p suppressed lung fibrosis by targeting Tab1

doi: 10.1038/s41598-023-29188-6

Figure Lengend Snippet: Osa-miR172d-5p identified as a plant miR candidate with an anti-fibrotic effect. ( A ) Scheme of plant miR selection. ( B ) In silico analysis of the interaction between osa-miR172d-5p and TAB1. ( C ) Human lung fibroblast HFL1 cells were transfected with the osa-miR172d-5p for 48 h, and TAB1 expression was evaluated via western blot analysis ( n = 4). ( D ) HFL1 cells were transfected with the indicated concentration of osa-miR172d-5p for 48 h, and cDNA was evaluated via qRT-PCR ( n = 4). Data are shown as mean ± SEM. * P < 0.05. *** P < 0.001 versus control group.

Article Snippet: Human lung fibroblast HFL1 cells (JCRB, Osaka, Japan) were cultured in 10% fetal bovine serum (FBS) (Sigma-Aldrich), Dulbecco's Modified Eagle Medium (DMEM) (044-29765; Fujifilm Tokyo, Japan) supplemented with penicillin G (876111)–streptomycin (876161; Meiji Pharmaceutical Co., Tokyo, Japan) in 5% CO 2 and 100% humidity at 37 °C.

Techniques: Selection, In Silico, Transfection, Expressing, Western Blot, Concentration Assay, Quantitative RT-PCR, Control

Journal: Scientific Reports

Article Title: Plant miRNA osa-miR172d-5p suppressed lung fibrosis by targeting Tab1

doi: 10.1038/s41598-023-29188-6

Figure Lengend Snippet: TAB1 knockdown suppressed TGFβ-induced fibrotic gene expression. ( A ) Human lung fibroblast HFL1 cells were transfected with TAB1-siRNA (10 nM, 48 h) and TAB1 expression levels were determined by western blot analysis. ( B – D ) Human lung fibroblast HFL1 cells were transfected with TAB1-siRNA (10 nM, 48 h) and treated with TGFβ (5 ng/mL for B , D 48 h; C 24 h). mRNA expression levels were assessed via RT-qPCR. ( B ) ASMA (αSMA) (n = 4), ( C ) COL1A1 (n = 4), and ( D ) FN (fibronectin; n = 4). Data are shown as mean ± SEM. * P < 0.05. ** P < 0.01. *** P < 0.001 versus control group.

Article Snippet: Human lung fibroblast HFL1 cells (JCRB, Osaka, Japan) were cultured in 10% fetal bovine serum (FBS) (Sigma-Aldrich), Dulbecco's Modified Eagle Medium (DMEM) (044-29765; Fujifilm Tokyo, Japan) supplemented with penicillin G (876111)–streptomycin (876161; Meiji Pharmaceutical Co., Tokyo, Japan) in 5% CO 2 and 100% humidity at 37 °C.

Techniques: Knockdown, Gene Expression, Transfection, Expressing, Western Blot, Quantitative RT-PCR, Control