human fetal lung fibroblast cell line (normal lung fibroblast, mrc-5 cells)  (Pasteur Institute)

Journal: Respiratory research

Article Title: Down-regulation of USP13 mediates phenotype transformation of fibroblasts in idiopathic pulmonary fibrosis.

doi: 10.1186/s12931-015-0286-3

Figure Lengend Snippet: Fig. 2 USP13 expression levels in primary fibroblast lines from IPF patients. a Graph of USP13 mRNA expression in primary IPF fibroblast lines (n = 5) and primary normal fibroblast lines (n = 3). USP13 mRNA expression levels were verified by real-time RT-PCR. b Protein expression levels of USP13 in the primary fibroblast lines were shown by western blot. c Graph of USP13 protein levels in representative primary fibroblast lines was shown. Relative intensity analysis of USP13 protein expression levels were assessed by fold-change of target protein/β-actin with standardization by setting the “normal control #1” as 1. β-actin was used as a control in both western blot and real-time RT-PCR assays. Error bars indicate the mean ± SEM. *p <0.05, **p <0.01

Article Snippet: One normal human fetal pulmonary fibroblast cell line (MRC-5) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Respiratory research

Article Title: Down-regulation of USP13 mediates phenotype transformation of fibroblasts in idiopathic pulmonary fibrosis.

doi: 10.1186/s12931-015-0286-3

Figure Lengend Snippet: Fig. 4 Proliferative, migratory, and invasive capacity of fibroblasts following the down-regulation of USP13. a Proliferation of cells with USP13 deficiency. At 48 h after transfection with USP13 siRNA (USP13si) or NC siRNA (NCsi), MRC-5 cell proliferation was assessed by a BrdU incorporation assay. The proliferating cells that recently underwent DNA synthesis were labeled with BrdU (green), and total nuclei were visualized by DAPI (blue) staining. % BrdU: Percent of BrdU+ cells within the siRNA-treated MRC-5 cell population. Error bars indicate the mean ± SEM. ***p <0.001. b The migratory and invasive capacity of fibroblasts with decreased USP13. Twenty-four hours after transfection with either USP13 siRNA or NC siRNA, equal numbers of fibroblasts were loaded into transwell chambers with or without matrigel. Images of migratory fibroblasts (top panel) and invasive fibroblasts (bottom panel) are shown from a crystal violet staining assay at different time points, as indicated

Article Snippet: One normal human fetal pulmonary fibroblast cell line (MRC-5) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transfection, BrdU Incorporation Assay, DNA Synthesis, Labeling, Staining

Journal: Respiratory research

Article Title: Down-regulation of USP13 mediates phenotype transformation of fibroblasts in idiopathic pulmonary fibrosis.

doi: 10.1186/s12931-015-0286-3

Figure Lengend Snippet: Fig. 5 The PTEN protein level in UPS13-deficient cells. a PTEN protein and mRNA levels in primary IPF fibroblasts. Relative intensity analysis of PTEN protein expression levels in primary IPF fibroblast lines (n = 5) and primary normal fibroblast lines (n = 3) were assessed by the fold-change of target protein/β-actin with standardization by setting the “normal control #1” as 1. PTEN mRNA expression was measured by real-time RT-PCR, and the fold-change was calculated by using the 2-ΔΔCT method. b, c MRC-5 cells were transfected with USP13 siRNA (USP13si) or NC siRNA (NCsi) for 48 h and PTEN protein (b) and mRNA (c) levels were measured by western blot and real-time RT-PCR. d MRC-5 cells were transfected with PTEN siRNA (PTENsi) or NCsi for 48 h, and USP13 protein expression was assessed by western blot. β-actin was used as a control in both the west- ern blot and real-time RT-PCR assays. Error bars indicate the mean ± SEM. *p <0.05, **p <0.01

Article Snippet: One normal human fetal pulmonary fibroblast cell line (MRC-5) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

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

Journal: Respiratory research

Article Title: Down-regulation of USP13 mediates phenotype transformation of fibroblasts in idiopathic pulmonary fibrosis.

doi: 10.1186/s12931-015-0286-3

Figure Lengend Snippet: Fig. 6 Levels of PTEN ubiquitination and degradation in USP13-deficient lung fibroblasts. a Endogenous USP13 was immunoprecipitated from MRC-5 whole cell lysates and immunoblotted with antibodies against USP13 and PTEN (left panel), while endogenous PTEN was immunoprecipitated from MRC-5 whole cell lysates and immunoblotted with antibodies against PTEN and USP13 (right panel). b Co-localization of USP13 and PTEN. PTEN (red) and USP13 (green) were labeled by immunofluorescence staining of MRC-5 cells. The right panel shows the overlay (merge) of PTEN, USP13, and nuclear DAPI staining (blue) in the same field to determine co-localization. Scale bar, 20 μm. c At 42 h after transfection with USP13 siRNA or NC siRNA, we added MG-132 (10 μM) for 6 h and harvested whole cell lysates. PTEN and ubiquitinated PTEN (Ub-PTEN) were assessed by immunoprecipitation and western blot. β-actin was used as a loading control. d Immunofluorescence staining of PTEN (red) and USP13 (green) in MRC-5 cells transfected with USP13 siRNA (USP13si) or negative control siRNA (NCsi) for 48 h. Nuclei were visualized by DAPI (blue) staining. Scale bar, 20 μm. (a–d) Data are representative of at least three independent experiments. e Representative serial lung sections from one IPF patient were stained for (a) H&E, (b) α-SMA, (c) PTEN, and (d) USP13. Images are representative of staining performed on samples from 11 individuals with IPF. Scale bar, 100 μm

Article Snippet: One normal human fetal pulmonary fibroblast cell line (MRC-5) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Labeling, Immunofluorescence, Staining, Transfection, Western Blot, Control, Negative Control