tissueculture vessels usingfibroblastbasal medium 2  (PromoCell)

Journal: PLOS ONE

Article Title: Quantitative imaging and semiotic phenotyping of mitochondrial network morphology in live human cells

doi: 10.1371/journal.pone.0301372

Figure Lengend Snippet: (A) Fluorescent representative imaging of nuclei using Hoechst staining (blue; HOE), membranes with CellMask dye (green; CMG) and cellular distribution of the mitochondria-specific dye MitoTracker Red FM (red; MTR) after irradiation with increasing doses of UVB (50 to 400 mJ/cm 2 ). Pictures were taken using live-cell confocal microscopy 6h after irradiation. Single, overlapping images and zoom inserts are presented. Scale bar = 10μm. (B) After analysis of a minimum of 300 cells with MitoTouch software, the distribution of normalized morphological features (Vn) is represented by a MitoSpider plot. Significant differences with respect to the control condition, n = 3 independent experiments, are notified by small (*: p<0.05), medium (**: p<0.01) or large (***: p<0.001) circles (t-test). Fibroblasts are sensitive to increasing UVB irradiation as shown by the purple broken lines in relation to the reference red circle (no UVB irradiation). Profiles are similar until 200 mJ/cm 2 , and slightly modified with 300 and 400 mJ/cm 2 as reported by the MitoSpider plots and the associated MitoSigils. (C) Projection of the samples onto the 2D space generated by the first two Principal Components (a). PCA2 segregates irradiated samples from untreated ones, independently of the irradiation dose. Correlation of each feature to the first two Principal Components (b). Projection of the samples onto the 3D space generated by the first three Principal Components (c). Projection of the samples onto the 2D space generated by the first two LDA discriminant axis (d). LDA1 splits irradiated samples from untreated ones, while LDA2 highlights the irradiation dose gradient. Correlation of each feature to the first two LDA discriminant axis (e). Note that features parallel to LDA1 have an explanatory role in the observed segregation between treated and untreated samples, whereas those parallel to LDA2 in the categorization of irradiated samples depending on their UVB dose. Projection of the samples onto the 3D space generated by the first three LDA discriminant axis (f). UVB doses are shown in the inset.

Article Snippet: During UVB irradiation and microscopy, NHDF were cultured in phenol-free Fibroblast Basal Medium-2 (C-23225) with supplementation of Growth Medium 2 SupplementMix (C-39325) and NHEK in phenol-free Keratinocyte Basal Medium (C-20216) and Growth Medium 2 SupplementMix (C-39016) (PromoCell, Germany).

Techniques: Imaging, Staining, Irradiation, Confocal Microscopy, Software, Control, Modification, Generated

Journal: International Journal of Molecular Sciences

Article Title: Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin

doi: 10.3390/ijms22168536

Figure Lengend Snippet: FENDRR has a negative effect on lung fibroblast proliferation. ( A ) Bright-field and GFP images of LL29 stable cells expressing GFP- FENDRR (FENDRR) and GFP vector control (VC). Scale bar: 100 µm. ( B ) Real-time PCR determination of FENDRR expression levels in the stable cells. FENDRR expression was normalized to β-actin and expressed as %VC. ( C ) Cell proliferation was determined by the BrdU assay in blank cells, VC, and FENDRR expressing stable cells after 6 days of culture. The results were expressed as %VC. ( D ) Nuclei staining of VC and FENDRR stable cells after 6 days of culture. Scale bar: 100 µm. ( E ) Cell counts of VC and FENDRR stable cells. In each experiment, 5 random fields were imaged, numbers of nuclei were counted, and cell counts were averaged. Cell counts were expressed as %VC. The cell counts for VC and FENDRR were 19.7 ± 2.0 and 11.3 ± 0.8 per field. Values represent the means ± SE. n = 3 independent experiments. * p < 0.05, ** p < 0.01 vs. VC. Student’s t -test for ( B , E ) and one-way ANOVA and Tukey’s multiple comparison for ( C ).

Article Snippet: HPFs were cultured in fibroblast medium (PromoCell, Cat. No: C-23220) with its supplements (PromoCell, Cat. No: C-39320) containing fetal calf serum (0.2 mL/mL), basic fibroblast growth factor (1 ng/mL), and insulin (5 μg/mL).

Techniques: Expressing, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, BrdU Staining, Staining, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin

doi: 10.3390/ijms22168536

Figure Lengend Snippet: FENDRR silencing increases lung fibroblast proliferation. ( A ) Bright-field and GFP images of HPF cells infected with FENDRR shRNA (sh FENDRR ) or shRNA control (shCon) lentiviruses (MOI 100 for 24 h). Scale bar: 100 µm. ( B ) Real-time PCR determination of FENDRR expression in HPF cells infected with sh FENDRR or shCon. FENDRR expression was normalized to β-actin and expressed as %shCon. ( C ) Cell proliferation was determined by the BrdU assay in blank cells and the cells infected with shCon and sh FENDRR after 6 days of culture. The results were expressed as %shCon. Values represent the means ± SE. n = 3 independent experiments. ** p < 0.01 vs. shCon. Student’s t -test for ( B ) and one-way ANOVA and Tukey’s multiple comparison for ( C ).

Article Snippet: HPFs were cultured in fibroblast medium (PromoCell, Cat. No: C-23220) with its supplements (PromoCell, Cat. No: C-39320) containing fetal calf serum (0.2 mL/mL), basic fibroblast growth factor (1 ng/mL), and insulin (5 μg/mL).

Techniques: Infection, shRNA, Control, Real-time Polymerase Chain Reaction, Expressing, BrdU Staining, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin

doi: 10.3390/ijms22168536

Figure Lengend Snippet: FENDRR reduces PS6K phosphorylation by binding SRSF9. ( A ) RNA immunoprecipitation showing the interaction of FENDRR with SRSF9 in LL29 cells. FENDRR enrichment was expressed as %IgG. ( B ) Western blot showing that FENDRR inhibits the phosphorylation of PS6K in LL29 stable cells expressing FENDRR or vector control (VC). ( C ) Quantitative analysis of protein levels of phosphorylated PS6K (p-PS6K). Values were normalized to total PS6K and then expressed as %VC. ( D ) Cell proliferation was determined by the BrdU assay at 6 days in LL29 cells infected with a lentivirus expressing a SRSF9 shRNA (MOI of 100 for 24 h). Values were expressed as %shCon. ( E ) Western blot showing the phosphorylation of PS6K at different times points (0, 15, 30, and 60 min) in LL29 cells treated with FGF2 (50 ng/mL). ( F ) Quantitative analysis of the phosphorylated PS6K and total PS6K protein levels from ( E ). Values were normalized to total PS6K and then expressed as % 0 Min. ( G ) Fibroblast proliferation in LL29 cells treated with FGF2 (50 ng/mL) for 3 days as determined by the BrdU assay. Values were expressed as %Blank. ( H ) Fibroblast proliferation in LL29 stable cells expressing vector control (VC) or FENDRR treated with FGF2 (80 ng/mL) for 3 days was determined by the BrdU assay. Values were expressed as %VC of Blank. Values represent the means ± SE. n = 3 independent experiments. * p < 0.05 vs. IgG, VC, or 0 min, ** p < 0.01 vs. Blank, **** p < 0.0001 vs. shCon, # p < 0.05 and ### p < 0.001 vs. VC. Student’s t -test for ( A , C , G ); one-way ANOVA and Tukey’s multiple comparison for ( D , F ); and two-way ANOVA and Tukey’s multiple comparison for ( H ).

Article Snippet: HPFs were cultured in fibroblast medium (PromoCell, Cat. No: C-23220) with its supplements (PromoCell, Cat. No: C-39320) containing fetal calf serum (0.2 mL/mL), basic fibroblast growth factor (1 ng/mL), and insulin (5 μg/mL).

Techniques: Phospho-proteomics, Binding Assay, RNA Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Control, BrdU Staining, Infection, shRNA, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin

doi: 10.3390/ijms22168536

Figure Lengend Snippet: β-catenin contributes to fibroblast proliferation. ( A ) XVA939 (10 µM)-mediated reduction of β-catenin protein levels in LL29 cells. The control is 0.1% DMSO. ( B ) Quantitative analysis of β-catenin protein levels in LL29 cells treated with XAV939. The results were expressed as %VC. ( C ) BrdU assay of cell proliferation in LL29 cells infected with adenovirus containing β-catenin shRNA constructs or treated with XVA939 for 6 days. The results were expressed as %shCon or %Control. ( D ) Western blot showing β-catenin levels in LL29 cells after transfection (nucleofection) of ΔGSK-β-catenin overexpressing plasmid or its control plasmid (CON). ( E ) Quantitative analysis of β-catenin protein levels in D. The results were normalized to β-actin and expressed as %CON. ( F ) The BrdU assay was performed to determine cell proliferation in LL29 stable cells expressing vector control (VC) or FENDRR at day 6 after transfecting ΔGSK-β-catenin overexpressing plasmid or its control plasmid (CON). The results were expressed as %VC of CON. Values represent the means ± SE. n = 3 independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. shCon, Control or VC. Student’s t -test for ( B , C , E ). Two-way ANOVA and Tukey’s multiple comparison for ( F ).

Article Snippet: HPFs were cultured in fibroblast medium (PromoCell, Cat. No: C-23220) with its supplements (PromoCell, Cat. No: C-39320) containing fetal calf serum (0.2 mL/mL), basic fibroblast growth factor (1 ng/mL), and insulin (5 μg/mL).

Techniques: Control, BrdU Staining, Infection, shRNA, Construct, Western Blot, Transfection, Plasmid Preparation, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin

doi: 10.3390/ijms22168536

Figure Lengend Snippet: A model showing that FENDRR inhibits fibroblast proliferation by binding SRSF9 and reducing β-catenin protein levels.

Article Snippet: HPFs were cultured in fibroblast medium (PromoCell, Cat. No: C-23220) with its supplements (PromoCell, Cat. No: C-39320) containing fetal calf serum (0.2 mL/mL), basic fibroblast growth factor (1 ng/mL), and insulin (5 μg/mL).

Techniques: Binding Assay