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

Journal: Journal of Biological Chemistry

Article Title: Calpain Is Required for the Rapid, Calcium-dependent Repair of Wounded Plasma Membrane

doi: 10.1074/jbc.m604560200

Figure Lengend Snippet: FIGURE 2. Calpastatin decreases calcium-dependent survival after scrap- ing. A, HFL1 human lung fibroblasts were scraped in the presence of calcium alone (C), Ca2/EGTA (E), calcium plus 20 M calpeptin (CP), or calcium plus 4 M calpastatin (CS). MTT reductase activity was measured after 6 h. *, p 0.001 versus all other experimental conditions (n 4). B, Chinese hamster ovary cells stably transfected with calpastatin under control of the insect ecdysonereceptorwereculturedfor4dayswithorwithout5Mponasterone A to induce calpastatin expression. The cells were scraped in the presence of calcium with or without 20 M calpeptin, and MTT reductase activity was measuredafter6h.*,p0.02versusallotherexperimentalconditions(n3).

Article Snippet: Human skin fibroblasts (ATCC CRL 1782) and HFL1 human lung fibroblasts (ATCCCRL153) were cultured in Iscove’s modified Dulbecco’s medium containing 10% FBS.

Techniques: Activity Assay, Stable Transfection, Transfection, Control, Expressing

Journal: Journal of Biological Chemistry

Article Title: Calpain Is Required for the Rapid, Calcium-dependent Repair of Wounded Plasma Membrane

doi: 10.1074/jbc.m604560200

Figure Lengend Snippet: FIGURE6.Calpainaccumulatedatthescrapeinjurysiteandwasactivated prior to vimentin cleavage. HFL1 human fibroblasts were scraped in the presence of calcium with or without the addition of 20 M calpeptin and biotinylated with MPB at various times thereafter as described under “Exper- imental Procedures.” After Neutravidin affinity gel purification, 10 g of pro- tein from each sample was electrophoresed, blotted, and immunostained with antibodies against vimentin (Vim), calpain-cleaved vimentin (vim frag), m-calpain large subunit, and calpain small subunit (SSU). *, relative migration of the 46-kDa calpain-generated vimentin fragment. NS, nonscraped.

Article Snippet: Human skin fibroblasts (ATCC CRL 1782) and HFL1 human lung fibroblasts (ATCCCRL153) were cultured in Iscove’s modified Dulbecco’s medium containing 10% FBS.

Techniques: Gel Purification, Migration, Generated

Journal: Journal of Biological Chemistry

Article Title: Calpain Is Required for the Rapid, Calcium-dependent Repair of Wounded Plasma Membrane

doi: 10.1074/jbc.m604560200

Figure Lengend Snippet: FIGURE 7. Distribution of calpain and calpastatin to the cell surface and cell fragment fractions after scrape injury. HFL1 cells were scraped and biotinylated at various times. The biotinylated proteins were affinity-purified on Neutravidin gel and subjected to SDS-PAGE in 10% polyacrylamide gels. The gels were either stained with Coomassie Blue (A) or electroblotted for immunologic studies (B). Lanes B, proteins purified by Neutravidin affinity chromatography. Lanes marked n, proteins that did not bind to the Neutravi- din gel. Lanes F, cell fragments present in the scraping medium after the 60-min labeling period with MPB were obtained in the supernatant of the first centrifugation step at 200 g (see “Experimental Procedures”). They were concentrated by sedimentation at 21,000 g and heated in 60 l of SDS sample buffer for electrophoretic analysis. Note that membrane fragments are observed in the nonscraped control fraction (NS). As stated under “Exper- imental Procedures,” these cells were not scraped prior to the addition of MPB, but they were scraped after the 60-min incubation on ice. Sample loads were as follows. A, 10 l/lane; B, 20 l/lane for lanes B and 5 l/lane for lanes N and F. ssu, small subunit.

Article Snippet: Human skin fibroblasts (ATCC CRL 1782) and HFL1 human lung fibroblasts (ATCCCRL153) were cultured in Iscove’s modified Dulbecco’s medium containing 10% FBS.

Techniques: Affinity Purification, SDS Page, Staining, Purification, Affinity Chromatography, Labeling, Centrifugation, Sedimentation, Membrane, Control, Incubation

Journal: Nature Communications

Article Title: c ircTP63 functions as a ceRNA to promote lung squamous cell carcinoma progression by upregulating FOXM1

doi: 10.1038/s41467-019-11162-4

Figure Lengend Snippet: Characterization of circTP63 in LUSC cells. a Genomic loci of circTP63 gene. circTP63 is produced at the TP63 gene (NM_003722.4) locus containing exons 10–11. The back-splice junction of circTP63 was identified by Sanger sequencing. b PCR analysis for circTP63 and its linear isoform TP63 in cDNA and genomic DNA (gDNA). c Northern blot analysis showed the size and abundance of circTP63 in one paired sample of LUSC tumorous tissue and corresponding adjacent nontumorous tissues. M: marker. d qRT-PCR for the abundance of circTP63 and TP63 in H1703 cells treated with Actinomycin D at the indicated time point. e Levels of circTP63 in the nuclear and cytoplasmic fractions of SW900 and H1703 cells. The error bars ( d , e ) represent s.d. ( n = 3)

Article Snippet: All of human LUSC cells (NCI-H2170, NCI-H1703, NCI-H226, NCI-H520, SW900, SK-MES-1, BEAS-2B, and HFL-1) were purchased from the American Type Culture Collection (ATCC) and were tested negative for mycoplasma contamination.

Techniques: Produced, Sequencing, Northern Blot, Marker, Quantitative RT-PCR

Journal: Nature Communications

Article Title: c ircTP63 functions as a ceRNA to promote lung squamous cell carcinoma progression by upregulating FOXM1

doi: 10.1038/s41467-019-11162-4

Figure Lengend Snippet: c ircTP63 promotes cell proliferation and tumor growth both in vitro and in vivo. a Expression levels of circTP63 and TP63 in SW900 and H1703 cells treated with circTP63 siRNA. b Expression levels of circTP63 and TP63 in H226 and H2170 cells after transduction with circTP63 lentivirus. c and d Cell proliferation analysis of LUSC cells with silencing or stably overexpressing circTP63 . e and f Cell cycle analysis of LUSC cells with silencing or stably overexpressing circTP63 . g The volume and weight of subcutaneous xenograft tumors of H2170 cells isolated from nude mice. h The volume and weight of subcutaneous xenograft tumors of H1703 cells isolated from nude mice; center line: median of data; Bounds of box: the second quartile to the third quartile; Whisker: minimum value to maximum value. The error bars a – h represent s.d. (in a – f , n = 3; in g and h , n = 6). * p < 0.05; ** p < 0.01; *** p < 0.001, two-tailed t -test. Source data are provided as a Source Data file

Article Snippet: All of human LUSC cells (NCI-H2170, NCI-H1703, NCI-H226, NCI-H520, SW900, SK-MES-1, BEAS-2B, and HFL-1) were purchased from the American Type Culture Collection (ATCC) and were tested negative for mycoplasma contamination.

Techniques: In Vitro, In Vivo, Expressing, Transduction, Stable Transfection, Cell Cycle Assay, Isolation, Whisker Assay, Two Tailed Test

Journal: Nature Communications

Article Title: c ircTP63 functions as a ceRNA to promote lung squamous cell carcinoma progression by upregulating FOXM1

doi: 10.1038/s41467-019-11162-4

Figure Lengend Snippet: c ircTP63 contributes to cell proliferation through targeting FOXM1. a Co-expression network of circTP63 with associated 25 mRNAs. A round node represents a protein-coding gene and the arrow node represents circTP63 ( hsa_circ_0068515 ). Lines between two nodes indicate interactions between two genes. Color represents the number of lines. b A heatmap shows mRNA levels of these 25 co-expression genes in the five paired LUSC samples of SBC Human ceRNA Array analysis. c Expression analysis for FOXM1 in additional 35 paired LUSC samples. d Correlation analysis revealed positive correlation between the levels of circTP63 and FOXM1 mRNA in the tumorous tissues of the 35 LUSC patients. ΔCt values were normalized according to β-actin . e The levels of circTP63 expression and FOXM1 protein in eight paired LUSC samples. f The mRNA and protein levels of FOXM1 in the LUSC cells with knockdown or overexpression of circTP63 . g Cell proliferation assay for H226 and H2170 cells with circTP63 overexpression and FOXM1 knockdown. h Cell proliferation assay for H1703 cells with circTP63 knockdown and FOXM1 overexpression. The error bars c , e – h represent s.d. (in c , n = 35; in e – h , n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001, two-tailed t -test. Source data are provided as a Source Data file

Article Snippet: All of human LUSC cells (NCI-H2170, NCI-H1703, NCI-H226, NCI-H520, SW900, SK-MES-1, BEAS-2B, and HFL-1) were purchased from the American Type Culture Collection (ATCC) and were tested negative for mycoplasma contamination.

Techniques: Expressing, Knockdown, Over Expression, Proliferation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: c ircTP63 functions as a ceRNA to promote lung squamous cell carcinoma progression by upregulating FOXM1

doi: 10.1038/s41467-019-11162-4

Figure Lengend Snippet: CENPA and CENPB are regulated by cicrTP63 through FOXM1. a The mRNA and protein levels of cell cycle-related genes in H226 and H2170 cells with circTP63 overexpression. b Expression changes of CENPA , CENPB , and CCNB1 after knockdown of FOXM1 in H2170 cells with circTP63 overexpression. c Cell proliferation assay for H226 and H2170 cells with circTP63 overexpression and joint knockdown of CENPA and CENPB . d Hypothesis diagram illustrates function and mechanism of circTP63 in LUSC progress. The error bars a – c represent s.d. ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001, two-tailed t -test. Source data are provided as a Source Data file

Article Snippet: All of human LUSC cells (NCI-H2170, NCI-H1703, NCI-H226, NCI-H520, SW900, SK-MES-1, BEAS-2B, and HFL-1) were purchased from the American Type Culture Collection (ATCC) and were tested negative for mycoplasma contamination.

Techniques: Over Expression, Expressing, Knockdown, Proliferation Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Lysionotin attenuates bleomycin-induced pulmonary fibrosis by activating AMPK/Nrf2 pathway

doi: 10.1038/s41598-025-17045-7

Figure Lengend Snippet: Lysionotin inhibits TGF-β-induced myofibroblast differentiation in 3 different cell types. The NIH3T3 cells, HFL-1 cells, and primary rat lung fibroblasts were incubated with different doses of Lysionotin and TGF-β (10 ng/mL) for 24 h. ( A ) The effects of the different concentrations of Lysionotin (0, 1, 3, 10, 30, and 100 µM) on cell viability were evaluated in NIH3T3 cells. ( B – E ) Western blotting and Q-PCR were used to detect the protein and mRNA expression levels of α-SMA and collagen I in NIH3T3 cells. ( F – J ) Cell viability and expression of α-SMA and collagen I (protein and mRNA) in HFL-1 cells. ( K – O ) Cell viability and expression of α-SMA and collagen I (protein and mRNA) in primary rat lung fibroblasts. The following notation is used: Lyn is the abbreviation of Lysionotin. Data are expressed as mean ± SD, n = 3, * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Human lung fibroblasts (HFL-1 cells) were purchased from Procell (Wuhan, China) and cultured with Ham’s F-12 K (Gibco, USA) containing 10% fetal bovine serum (Cat# F2442, FBS, Sigma, USA) and 1% penicillin–streptomycin solution.

Techniques: Incubation, Western Blot, Expressing

Journal: Molecular cancer therapeutics

Article Title: TAS-116, a novel Hsp90 inhibitor, selectively enhances radio-sensitivity of human cancer cells to X-rays and carbon ion radiation

doi: 10.1158/1535-7163.MCT-16-0573

Figure Lengend Snippet: TAS-116 sensitizes human cancer cells to radiation. HeLa, H1299, and HFL1 cells were pretreated with 1 µM TAS-116 for 24 hr, and irradiated with X rays (A) or carbon ions (B). Colonies with more than 50 cells were scored. Data represent mean ± SEM of two independent experiments. Asterisks indicate significant differences between TAS-116 treated and mock treated cells (Student’s t-test, p < 0.05).

Article Snippet: HFL1 human fibroblasts were purchased from RIKEN BioResource Center in 2002 and cultured in alpha MEM supplemented with 15% FBS.

Techniques: Irradiation

Journal: bioRxiv

Article Title: Circular RNA RORβ regulates TGFBR1by decoying miR-140 in alcohol-exposed lungs and fibroblasts

doi: 10.1101/2022.05.27.492519

Figure Lengend Snippet: (A) Human lung fibroblasts (HLF) were treated with alcohol (ALC, 60 mM, 48h). In parallel, (B) murine primary lung fibroblasts (MLF) were treated with alcohol (ALC, 60 mM, 48h). (C) Alcohol-fed mice were sacrificed after 8 weeks of alcohol ingestion (ALC; 20% v/v of ethanol in drinking water, ALC) along with their control-fed counterparts (CON). Samples were harvested and analyzed for TGFβ receptor type 1 (TGFβR1) expression by qPCR. N=4-7. *p<0.05.

Article Snippet: Normal human lung fibroblasts (HLF) were obtained from ATCC (CCL-153) and were cultured in F-12K medium supplemented with 10% FBS per manufacturer protocol.

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: Circular RNA RORβ regulates TGFBR1by decoying miR-140 in alcohol-exposed lungs and fibroblasts

doi: 10.1101/2022.05.27.492519

Figure Lengend Snippet: (A) Human lung fibroblasts (HLF) were treated with alcohol (ALC, 60 mM, 48h). In parallel, (B) murine primary lung fibroblasts (MLF) were treated with alcohol (ALC, 60 mM, 48h). (C) Alcohol-fed mice were sacrificed after 8 weeks of alcohol ingestion (ALC; 20% v/v of ethanol in drinking water, ALC) along with their control-fed counterparts (CON). Samples were harvested and analyzed for circ-RORβ expression by qPCR. N=4-6. *p<0.05.

Article Snippet: Normal human lung fibroblasts (HLF) were obtained from ATCC (CCL-153) and were cultured in F-12K medium supplemented with 10% FBS per manufacturer protocol.

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: Circular RNA RORβ regulates TGFBR1by decoying miR-140 in alcohol-exposed lungs and fibroblasts

doi: 10.1101/2022.05.27.492519

Figure Lengend Snippet: (A) Image shows the top 10 miRNAs which could interact with both circ-RORβ and TGFβR1. (B) Human lung fibroblasts (HLF) were treated with alcohol (ALC, 60 mM, 48h), then analyzed for miR-143, miR-144, miR-153, miR-330, miR-330, miR-338, miR-409, miR-432, miR-490, and miR-888 by qPCR. (C) alcohol-treated HLF (ALC, 60 mM, 48h) were analyzed for miR-140 expression by qPCR. In parallel, (D) alcohol-treated murine primary lung fibroblasts (MLF) (ALC, 60 mM, 48h) and (E) the lung from alcohol-fed mice (ALC; 20% v/v of ethanol in drinking water, ALC) were analyzed for miR-140 expression by qPCR. N=3-7. *p<0.05.

Article Snippet: Normal human lung fibroblasts (HLF) were obtained from ATCC (CCL-153) and were cultured in F-12K medium supplemented with 10% FBS per manufacturer protocol.

Techniques: Expressing

Journal: bioRxiv

Article Title: Circular RNA RORβ regulates TGFBR1by decoying miR-140 in alcohol-exposed lungs and fibroblasts

doi: 10.1101/2022.05.27.492519

Figure Lengend Snippet: Human LFs (HLF) were transfected with synthetic anti-sense oligonucleotides against circ-RORβ (ASO circ-RORβ; 50 nM) or scrambled ASO (50 nM) using lipofectamine 3000. At 24 hours following transfection, cells were treated without (CON) or with alcohol (ALC; 60 mM) for an additional 48 hours then analyzed for (A) miR-140 expressions, (B) TGFβR1 and markers for FMD including (C) fibronectin, and (D) αSMA expression by qPCR. In parallel, cells were maintained in media with or without alcohol (60 mM) for an additional 96 hours following ASO circ-RORβ transfection, then fixed and stained with rabbit anti-human αSMA (green) and DAPI (blue). Cells treated with recombinant human TGFβ1 (5 ng/ml) were used as a positive control. Cells were counted from 3 random 20x field. Total cells counted of 46-215. (E) Graph depicts percent of cells with stress fiber formation to total cells. Representative slide from untreated (CON), TGFβ1, scramble AOS-transfected followed by alcohol-treated (ALC+ASO SCR), and ASO circ-RORβ-transfected followed by alcohol-treated (ALC+ASO Circ-RORβ) were shown, 40x magnification. *p<0.05 compared to untreated group. +p<0.05 compared to alcohol-treated group. (F) Diagram summarizes the findings presented in this study. In lung fibroblasts, alcohol induces circ-RORβ expression which, in turn, neutralizes miR-140. This interaction leads to an upregulation of TGFβR1 expression in alcohol-treated lung fibroblasts. These sequential events promote fibroblasts to differentiate into myofibroblasts which underlies the susceptibility to injury and disrepair in alcoholic lungs.

Article Snippet: Normal human lung fibroblasts (HLF) were obtained from ATCC (CCL-153) and were cultured in F-12K medium supplemented with 10% FBS per manufacturer protocol.

Techniques: Transfection, Expressing, Staining, Recombinant, Positive Control