Journal: Scientific Reports

Article Title: TAZ contributes to pulmonary fibrosis by activating profibrotic functions of lung fibroblasts

doi: 10.1038/srep42595

Figure Lengend Snippet: ( a ) siRNA-transfected HFL-1 cells were stained with fluorescein-phalloidin (green) to visualize F-actin. DAPI was used for nuclear staining (blue). Scale bar = 100 μm. ( b ) siRNA-transfected HFL-1 cells were seeded at a density of 2 × 10 4 /well in quadruplicate. Cell numbers were counted after 24, 72, and 120 h. ( c ) Migration of siRNA-transfected HFL-1 cells was measured with the Boyden blind-well assay system (n = 8 for each group). Human fibronectin was added to the lower chambers as a chemoattractant. The cells were seeded onto the upper chambers (8 × 10 4 /well) and allowed to migrate for 6 h. The number of migrated cells was determined by the WST-8 assay. ( d ) siRNA-transfected HFL-1 cells were embedded in collagen gels. After 72 h, the area of each gel was quantified. Representative photographs of collagen gels (upper). Percentage of contracted gel area compared to the initial size (lower). Error bars represent standard deviations. * P < 0.05, Student’s t -test.

Article Snippet: Cells were fixed in formalin, permeabilized with 0.3% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. The cells were incubated with anti-TAZ antibody (1:250 dilution, HPA007415; Sigma-Aldrich) or anti-α-SMA (1:250 dilution, Sigma-Aldrich) at 4 °C overnight, and further incubated for 1 h with secondary goat anti-rabbit Alexa Fluor 594-conjugated antibody (1:500 dilution, Life Technologies) or anti-mouse Alexa Fluor 488-conjugated antibody (1:500 dilution, Life Technologies), respectively. siRNA-transfected HFL-1 cells were stained with Acti-stain 488 phalloidin to detect F-actin (Cytoskeleton Inc., Denver, CO, USA), and the nuclei were stained with DAPI.

Techniques: Transfection, Staining, Migration

Journal: Scientific Reports

Article Title: TAZ contributes to pulmonary fibrosis by activating profibrotic functions of lung fibroblasts

doi: 10.1038/srep42595

Figure Lengend Snippet: HFL-1 cells were seeded on type I collagen-coated polyacrylamide hydrogels with different stiffness values (0.5 and 25 kPa). TAZ expression was examined after 48 h by immunofluorescence (red). Cells were also stained with fluorescein-phalloidin (green) to visualize F-actin. DAPI was used for nuclear staining (blue). ( a ) Low magnification. Scale bar = 50 μm. ( b ) High magnification of the area in the white box in ( a ). Scale bar = 10 μm.

Article Snippet: Cells were fixed in formalin, permeabilized with 0.3% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. The cells were incubated with anti-TAZ antibody (1:250 dilution, HPA007415; Sigma-Aldrich) or anti-α-SMA (1:250 dilution, Sigma-Aldrich) at 4 °C overnight, and further incubated for 1 h with secondary goat anti-rabbit Alexa Fluor 594-conjugated antibody (1:500 dilution, Life Technologies) or anti-mouse Alexa Fluor 488-conjugated antibody (1:500 dilution, Life Technologies), respectively. siRNA-transfected HFL-1 cells were stained with Acti-stain 488 phalloidin to detect F-actin (Cytoskeleton Inc., Denver, CO, USA), and the nuclei were stained with DAPI.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: TAZ contributes to pulmonary fibrosis by activating profibrotic functions of lung fibroblasts

doi: 10.1038/srep42595

Figure Lengend Snippet: ( a ) Gene Ontology analysis of the commonly downregulated genes in HFL-1 cells transfected with TAZ siRNA #1 or #2. The data presented is log transformed P -values of GO terms enriched in the TAZ-regulated genes. ( b ) Relative expression levels of the indicated transcripts in HFL-1 and WI-38 cells transfected with siNTC or siTAZ were analyzed by qRT-PCR. Error bars represent standard deviations. * P < 0.05, Student’s t -test. ( c ) Immunoblotting for TAZ and YAP, and CTGF in HFL-1 cells transfected with TAZ siRNA #1 and/or YAP siRNA in the presence or absence of 5 ng/ml TGF-β stimulation for 48 h. GAPDH was used as the loading control. Molecular weight of each protein is indicated.

Article Snippet: Cells were fixed in formalin, permeabilized with 0.3% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. The cells were incubated with anti-TAZ antibody (1:250 dilution, HPA007415; Sigma-Aldrich) or anti-α-SMA (1:250 dilution, Sigma-Aldrich) at 4 °C overnight, and further incubated for 1 h with secondary goat anti-rabbit Alexa Fluor 594-conjugated antibody (1:500 dilution, Life Technologies) or anti-mouse Alexa Fluor 488-conjugated antibody (1:500 dilution, Life Technologies), respectively. siRNA-transfected HFL-1 cells were stained with Acti-stain 488 phalloidin to detect F-actin (Cytoskeleton Inc., Denver, CO, USA), and the nuclei were stained with DAPI.

Techniques: Transfection, Transformation Assay, Expressing, Quantitative RT-PCR, Western Blot, Molecular Weight

Journal: Scientific Reports

Article Title: TAZ contributes to pulmonary fibrosis by activating profibrotic functions of lung fibroblasts

doi: 10.1038/srep42595

Figure Lengend Snippet: ( a ) Correlation between TAZ expression level and percent predicted forced vital capacity (FVC) or diffusing capacity of the lungs for carbon monoxide (DLCO) in patients with ILD. TAZ expression data were extracted from the mRNA expression catalogue available in the Lung Genomic Research Consortium (LGRC) database (Probe set: A_23_P29769). Details about the samples are available at the LGRC website ( https://www.lung-genomics.org ). Spearman correlation coefficients (rho) and P -values were calculated (n = 249 and 221, respectively). ( b ) Gene set enrichment analysis (GSEA) was performed in the LGRC cohort using the set of 94 TAZ-regulated genes in HFL-1 cells. Patients with ILD were divided into two groups according to percent predicted FVC (cutoff, 50%). The enrichment of TAZ-regulated genes is shown schematically with the genes that correlated best with low FVC on the left and those that correlated best with high FVC on the right.

Article Snippet: Cells were fixed in formalin, permeabilized with 0.3% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. The cells were incubated with anti-TAZ antibody (1:250 dilution, HPA007415; Sigma-Aldrich) or anti-α-SMA (1:250 dilution, Sigma-Aldrich) at 4 °C overnight, and further incubated for 1 h with secondary goat anti-rabbit Alexa Fluor 594-conjugated antibody (1:500 dilution, Life Technologies) or anti-mouse Alexa Fluor 488-conjugated antibody (1:500 dilution, Life Technologies), respectively. siRNA-transfected HFL-1 cells were stained with Acti-stain 488 phalloidin to detect F-actin (Cytoskeleton Inc., Denver, CO, USA), and the nuclei were stained with DAPI.

Techniques: Expressing

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: 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: 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