Journal: The Journal of biological chemistry

Article Title: The novel type II prolyl 4-hydroxylase is the main enzyme form in chondrocytes and capillary endothelial cells, whereas the type I enzyme predominates in most cells.

doi: 10.1074/jbc.273.11.5989

Figure Lengend Snippet: FIG. 1. Contributions of the two isoenzymes to total prolyl 4-hydroxylase activity in cultured cells. Black columns represent type I and hatched columns type II as percentages of total prolyl 4-hydroxylase activity in logarithmic phase (L) and confluent (C) cells. The cell lines studied are adult human skin fibroblasts (AHSF), fetal human skin fibroblasts (FHSF), mouse embryonal fibroblasts (3T3), mouse chondrocytes (MC), human embryonic lung fibroblasts (WI-38), simian virus 40-transformed WI-38 cells (Va-13), and human embryo- nal rhabdomyosarcoma cells (RD). The results are given as the means from two to six separate experiments and their S.D. values or ranges (in the cases of WI-38 cells and logarithmic phase fetal human skin fibro- blasts and Va-13 cells with n 5 2).

Article Snippet: Cell Cultures—The cell lines used here were fetal human skin fibroblasts (ATCC CRL-1475), adult human skin fibroblasts (ATCC CRL1987), human embryonic lung fibroblasts (WI-38, ATCC CCL-75), simian virus 40-transformed WI-38 cells (Va13/WI-38, ATCC CCL 75.1), human embryonal rhabdomyosarcoma cells (RD, ATCC CCL 136), mouse embryonal fibroblasts (3T3, ATCC CCL-92), and mouse chondrocytes, which were obtained from the heads of the ribs of 7-day-old mice.

Techniques: Activity Assay, Cell Culture, Virus, Transformation Assay

Journal: The Journal of biological chemistry

Article Title: The novel type II prolyl 4-hydroxylase is the main enzyme form in chondrocytes and capillary endothelial cells, whereas the type I enzyme predominates in most cells.

doi: 10.1074/jbc.273.11.5989

Figure Lengend Snippet: FIG. 3. Immunofluorescence staining of samples from a fetal human foot with antibodies to prolyl 4-hydroxylase a(I) subunit (A, C, E, and G) and a(II) subunit (B, D, F, and H). A and B, undifferentiated mesenchymal cells are stained strongly with the a(I) antibody (A) but show only a very weak staining with the a(II) antibody (B). Capillaries (arrows) around the mesenchymal cells show strong immunofluorescence with the a(II) antibody (B). C and D, chondrocytes show intensive staining with both a(I) (C) and a(II) (D) antibodies. The synovial cells covering the joints stain strongly with the a(I) antibody (C) and much less so with the a(II) antibody (D). E and F, a strong signal with the a(I) antibody is seen in osteoblasts (arrows) (E), while a weaker but clear signal is seen with the a(II) antibody (F). G, epidermal cells (ep) and dermal (d) fibroblasts show strong staining with the a(I) antibody, whereas the dermal capillaries (arrows) are negative. H, the endothelial cells of the capillaries demonstrate high positivity for the a(II) antibody (arrows), whereas the epidermal cells (ep) and dermal (d) fibroblasts give no signal. The basement membrane below the epider- mis shows a weak staining. Magnification: 3 100, except 3 160 in G.

Article Snippet: Cell Cultures—The cell lines used here were fetal human skin fibroblasts (ATCC CRL-1475), adult human skin fibroblasts (ATCC CRL1987), human embryonic lung fibroblasts (WI-38, ATCC CCL-75), simian virus 40-transformed WI-38 cells (Va13/WI-38, ATCC CCL 75.1), human embryonal rhabdomyosarcoma cells (RD, ATCC CCL 136), mouse embryonal fibroblasts (3T3, ATCC CCL-92), and mouse chondrocytes, which were obtained from the heads of the ribs of 7-day-old mice.

Techniques: Immunofluorescence, Staining, Membrane

Journal: Cancer Science

Article Title: Elevated expression of angiomodulin (AGM/IGFBP‐rP1) in tumor stroma and its roles in fibroblast activation

doi: 10.1111/j.1349-7006.2012.02203.x

Figure Lengend Snippet: Effect of transforming growth factor‐β1 (TGF‐β1) on expression of angiomodulin (AGM), fibronectin (FN) and α‐smooth muscle actin (α‐SMA) in two kinds of cultured human fibroblasts. Human natal dermal fibroblasts (HDFs) (a) and WI38 cells (b) were incubated with the indicated concentrations (ng/mL) of TGF‐β1 in serum‐free medium for 2 days. From each culture, the conditioned medium and cell lysates were prepared, as described in Materials and Methods. AGM and fibronectin were analyzed with the conditioned media, while α‐SMA and β‐actin as an internal loading control were done with the cell lysates. The results were reproduced in at least three separate experiments.

Article Snippet: Human fetal lung fibroblast cell line WI38 and human bladder carcinoma cell line T24 (EJ‐1 strain) were provided from Japanese Collection of Research and Bioresources (JCRB, Osaka, Japan).

Techniques: Expressing, Cell Culture, Incubation, Control

Journal: Cancer Science

Article Title: Elevated expression of angiomodulin (AGM/IGFBP‐rP1) in tumor stroma and its roles in fibroblast activation

doi: 10.1111/j.1349-7006.2012.02203.x

Figure Lengend Snippet: Effects of angiomodulin (AGM) and transforming growth factor‐β1 (TGF‐β1) on growth of human fibroblasts. (a,b) Human natal dermal fibroblasts (HDFs) were incubated with the indicated concentrations of TGF‐β1 for 5 days (a) or AGM for 4 days (β) in DMEM/F12+5% FCS medium on 24‐well plates. Each point represents the mean ± SD of the numbers of cells in triplicate wells. (c) Time course of HDF growth in presence (●) or absence (○) of 10 μg/mL AGM. (d) Effect of varied concentrations of AGM on the growth of HDFs was examined in the presence (●) or absence (○) of 10 μM Smad inhibitor SB431542 (Smad inh.) for 6 days on a 96‐well plate. The cell growth was measured by the crystal violet staining. Each point represents the mean ± SD in triplicate wells. (e,f) Effects of varied concentrations of TGF‐β1 (e) or ΑGΜ (f) on the growth of WI38 cells were examined for 5 days as described in (a) and (b). Other experimental conditions are described in Materials and Methods.

Article Snippet: Human fetal lung fibroblast cell line WI38 and human bladder carcinoma cell line T24 (EJ‐1 strain) were provided from Japanese Collection of Research and Bioresources (JCRB, Osaka, Japan).

Techniques: Incubation, Staining

Journal: Cancer Science

Article Title: Miconazole inhibits signal transducer and activator of transcription 3 signaling by preventing its interaction with DNA damage‐induced apoptosis suppressor

doi: 10.1111/cas.14432

Figure Lengend Snippet: Miconazole (MIC) inhibits cell growth, migration and invasion of lung cancer. A, Western blot analysis in human normal lung fibroblast cells (WI‐38 and IMR‐90) and non–small‐cell lung carcinoma (NSCLC) (H23, H1703, H1793 and H2009). B, Cell growth of WI‐38, H23, H1703, H1793 or H2009 cells determined by SRB assay following culturing for 72 h after MIC treatment. C, Annexin V staining of cells treated with MIC (10 µmol/L) for 72 h. D, Western blot analysis of cells treated with MIC (10 µmol/L) for 72 h. E, Migration assay of cells treated with MIC for 12 h. F, Invasion assay of cells treated with MIC for 24 h. The values represent means ± SEM. ** P < 0.01, *** P < 0.001

Article Snippet: Human embryonic kidney cells expressing the SV40 large T antigen (HEK293T), human lung fibroblasts (WI‐38 and IMR‐90), and non–small cell lung cancer cell lines (NCI‐H23, NCI‐H1703, NCI‐H1793 and NCI‐H2009) were purchased from the ATCC or the Korean Cell Line Bank.

Techniques: Migration, Western Blot, Sulforhodamine B Assay, Staining, Invasion Assay

Journal: Scientific Reports

Article Title: Transcriptome analysis of periodontitis-associated fibroblasts by CAGE sequencing identified DLX5 and RUNX2 long variant as novel regulators involved in periodontitis

doi: 10.1038/srep33666

Figure Lengend Snippet: ( a ) Hierarchical clustering analysis with all DPIs of 6 gingival fibroblasts (GFs) (red), 6 periodontal ligament fibroblasts (PLFs) (blue), and 33 other fibroblasts derived from different anatomic sites (green) by Ward’s method. The details of fibroblasts are described in . Red to yellow color gradient of heatmap represents the degree of correlation of the indicated cell pair. ( b ) MA plot showing expression differences between GFs and 33 other fibroblasts. Red marks indicate promoters with significantly differential expression defined by false discovery rate (FDR) < 0.05, and blue marks indicate the genes with highly differential expression after sorting by FDR. The x -axis represents expression strength of a gene measured by CAGE tag counts and shown as average log 2 counts per million. The y -axis represents fold changes of gene expression shown as log 2 values. Positive fold changes indicate higher expression in GFs. ( c ) Venn diagram of genes with higher expression in GFs compared to other fibroblasts. The results from 3 microarray datasets were merged (GSE3551, GSE19090, GSE22029). The number of genes with higher expression in GFs (log 2 fold change >1, FDR < 0.05) is indicated in parenthesis. The indicated 9 genes were common in the 3 datasets. Among them, 5 genes highlighted in red were also enriched in GFs as determined by CAGE profiling ( b ). ( d ) RT-qPCR for BARX1 , LHX8 , and PAX9 in GFs (black bars: GF4 and GF5), dermal fibroblasts (DF, grey bar: NB1RGB), and lung fibroblasts (LFs, white bars: NHLF, WI38, and HFL1). The expression of each gene was normalized to that of GAPDH . Bars represent mean ± SD. No CT indicates the failure to calculate Ct (cycle threshold) values due to undetectable expression. ( e ) RT-qPCR for MEIS1 and HOXB2 in GFs, DF, and LFs. The data is presented as in ( d ).

Article Snippet: Human fetal lung fibroblasts (HFL1 and WI38) and adult normal human lung fibroblasts (NHLF) were obtained from American Type Culture Collection (ATCC) and Lonza (Basel, Switzerland), respectively.

Techniques: Derivative Assay, Expressing, Quantitative Proteomics, Gene Expression, Microarray, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Transcriptome analysis of periodontitis-associated fibroblasts by CAGE sequencing identified DLX5 and RUNX2 long variant as novel regulators involved in periodontitis

doi: 10.1038/srep33666

Figure Lengend Snippet: ( a ) CAGE peaks of 6 GFs and 33 other fibroblasts visualized by the ZENBU browser. Genomic coordinate and transcript of DLX5 registered in RefSeq (NM_005221) are shown on the top. CAGE peak for p1 DLX5 is prominent in GFs in contrast to 33 other fibroblasts. ( b ) CAGE peaks of 6 GFs and 33 other fibroblasts visualized by the ZENBU browser. Genomic coordinate and 3 protein coding transcript variants of RUNX2 registered in RefSeq (NM_004348, NM_001015051, and NM_001024630) are shown on the top. CAGE peaks for both p1 RUNX2 and p2 RUNX2 are detected in GFs while other fibroblasts show a dominant peak for p2 RUNX2. ( c ) RT-qPCR for DLX5 , RUNX2 long form transcribed from p1 RUNX2 promoter, and RUNX2 short form transcribed from p2 RUNX2 promoter. Primers for RUNX2 long form were designed so that one half hybridizes to the second exon and the other half to the third exon. Primers for RUNX2 short form were designed to amplify the isoform-specific sequence of the first exon. The expression of each gene was normalized to that of GAPDH . Bars represent mean ± SD.

Article Snippet: Human fetal lung fibroblasts (HFL1 and WI38) and adult normal human lung fibroblasts (NHLF) were obtained from American Type Culture Collection (ATCC) and Lonza (Basel, Switzerland), respectively.

Techniques: Quantitative RT-PCR, Sequencing, Expressing

Journal: Scientific Reports

Article Title: Transcriptome analysis of periodontitis-associated fibroblasts by CAGE sequencing identified DLX5 and RUNX2 long variant as novel regulators involved in periodontitis

doi: 10.1038/srep33666

Figure Lengend Snippet: ( a ) RNA-seq of GF8 (lower panel) and CAGE peak of GF4 (upper panel). LHX8 and neighboring ncRNAs which are specifically expressed in normal GFs were visualized by the ZENBU browser. ( b ) RT-qPCR for lnc-LHX8 in GFs (GF4, GF5, GF7, and GF8) and other fibroblasts (DF and LFs). The expression was normalized to that of GAPDH . Bars represent mean ± SD.

Article Snippet: Human fetal lung fibroblasts (HFL1 and WI38) and adult normal human lung fibroblasts (NHLF) were obtained from American Type Culture Collection (ATCC) and Lonza (Basel, Switzerland), respectively.

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: Transcriptome analysis of periodontitis-associated fibroblasts by CAGE sequencing identified DLX5 and RUNX2 long variant as novel regulators involved in periodontitis

doi: 10.1038/srep33666

Figure Lengend Snippet: ( a ) Collagen gels embedded with patient-matched periodontitis-associated fibroblasts (PAFs) or non-PAFs derived from healthy gingival tissues. Gingival epithelial cells, which are not patient-matched, were co-cultured on the surface of each gel for 5 d. Representative pictures of collagen gels cultured separately under air-liquid interface conditions. The brown spot in each well is the remaining collagen gel. The size of collagen gel embedded with PAFs was smaller than that seen in the non-PAF treatment. ( b ) Representative pictures of hematoxylin and eosin (H&E) staining of collagen gels embedded with PAFs or non-PAFs. Arrows indicate degradation of collagen gel matrix adjacent to PAFs. Bar represents 25 μm.

Article Snippet: Human fetal lung fibroblasts (HFL1 and WI38) and adult normal human lung fibroblasts (NHLF) were obtained from American Type Culture Collection (ATCC) and Lonza (Basel, Switzerland), respectively.

Techniques: Derivative Assay, Cell Culture, Staining