Journal: International Journal of Medical Sciences

Article Title: High Efficiency Low Cost Fibroblast Nucleofection for GMP Compatible Cell-based Gene Therapy

doi: 10.7150/ijms.19241

Figure Lengend Snippet: Analysis of transfection efficiency of rat dermal fibroblasts. Rat fibroblasts were isolated, shortly cultured (passage number did not exceed 3) and transfected with pmaxGFP plasmid. Transfection efficiency was analyzed by flow cytometry of GFP expression and was given as the percentage of GFP positive cells. A) Phenotypical characterization of rat dermal fibroblasts. The cells were evaluated with phalloidin/DAPI staining (upper panel) as well as by staining with rat fibroblast-specific antibody against beta subunit of prolyl-4-hydroxilase and propidium iodide (lower panel). B) Comparison of the transfection efficiencies of the four different non-viral transfection methods. Images of light and fluorescent microscopy are given in the upper panel and GFP transfected cells in the lower panel. C) Comparison of the transfection efficiencies of standard and modified nucleofection protocol (standard transfection solution was substituted with DMEM cell culture medium supplemented with 10% FCS). Images of light and fluorescent microscopy are given in the upper panel and GFP transfected cells in the lower panel. D) Time-course of the percentage of GFP positive fibroblasts transfected by using the modified nucleofection protocol. Images of fluorescent microscopy are given in the upper panel. Scale bar represents 100 μm in A upper panel, 50 μm in lower panel and 200 μm in others. The results are depicted as mean ± SD, t-test: *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: After cell isolation, four common non-viral transfection methods were used for the transfection of rat dermal fibroblasts: 1) Lipofectamine 2000 (Invitrogen, California, USA), 2) Jet PEI (Polyplus-transfection SA, Strasbourg, France), 3) Calcium Phosphate Transfection Kit (Invitrogen, California, USA) and 4) Transfection with the Nucleofector apparatus (later in the text referred as nucleofection) by using the Nucleofactor Kit for primary mammalian fibroblasts as described by the manufacturer (Lonza, Cologne, Germany).

Techniques: Transfection, Isolation, Cell Culture, Plasmid Preparation, Flow Cytometry, Expressing, Staining, Comparison, Microscopy, Modification

Journal: International Journal of Medical Sciences

Article Title: High Efficiency Low Cost Fibroblast Nucleofection for GMP Compatible Cell-based Gene Therapy

doi: 10.7150/ijms.19241

Figure Lengend Snippet: Analysis of transfection efficiency of human dermal fibroblasts. Human fibroblasts were isolated from split skin, cultured (passage number did not exceed 3) and transfected with pmaxGFP plasmid. Transfection efficiency was analyzed by flow cytometry of GFP expression and was given as the percentage of GFP positive cells. A) Phenotypical characterization of human dermal fibroblasts. The cells were evaluated with phalloidin/DAPI staining (upper panel) as well as by staining with human fibroblast-specific antibody against Thy-1 and propidium iodide (lower panel). (B) Transfection efficiency of three transfection solutions was compared by measuring the GFP expression (lower panel). Images of fluorescent microscopy are given in the upper panel. C) Time-course of the percentage of GFP positive fibroblasts transfected by using the modified nucleofection protocol and ITS liquid media supplement (right panel). Images of fluorescent microscopy are given in the left panel. Scale bar represents 100 μm in A upper panel, 50 μm in lower panel and 200 μm in others. The results are depicted as mean ± SD, t-test: ***p<0.001.

Article Snippet: After cell isolation, four common non-viral transfection methods were used for the transfection of rat dermal fibroblasts: 1) Lipofectamine 2000 (Invitrogen, California, USA), 2) Jet PEI (Polyplus-transfection SA, Strasbourg, France), 3) Calcium Phosphate Transfection Kit (Invitrogen, California, USA) and 4) Transfection with the Nucleofector apparatus (later in the text referred as nucleofection) by using the Nucleofactor Kit for primary mammalian fibroblasts as described by the manufacturer (Lonza, Cologne, Germany).

Techniques: Transfection, Isolation, Cell Culture, Plasmid Preparation, Flow Cytometry, Expressing, Staining, Microscopy, Modification

Journal: PLoS ONE

Article Title: Cytoskeletal alteration modulates cancer cell invasion through RhoA-YAP signaling in stromal fibroblasts

doi: 10.1371/journal.pone.0214553

Figure Lengend Snippet: (A) Expression of αSMA and vimentin in fibroblasts (hTERT-hNOFs and CAFs) mono-cultured and co-cultured with OSCC YD10B cells for 48 h. Cell lysates were prepared, and then Western blot were performed. (B) Cells grown in the presence or absence of YD10B OSCC cells. After 24h, the cells were harvested and detected mRNA expression of IL-6 and IL-8. (C) After 24h, conditioned medium was collected and the secretion levels of IL-6 and IL-8were measured by ELISA. The results were normalized for total protein and expressed as pg/mg of protein. This assay was performed in independent triplicate repeats (* p < 0.05; Mann-Whitney U test). (D) Representative images are shown to observe OSCC cells (YD10B and YD38) invaded toward stromal proportion with or without fibroblasts (hTERT-hNOFs and CAF#1) in 3D culture conditions. The mixture of each fibroblast and collagen was put into Mill-cell and grown for 14 days. Thereby, the rafts were fixed, embedded in paraffin, sectioned and stained using hematoxylin and eosin. (200x magnification, Scale bar: 100 μm, respectively). (E) SA-β-Gal assay was performed and representative microscopic pictures (a) mono-cultured hTERT-hNOFs (left, top), (d) hTERT-hNOFs, co-cultured with YD10B OSCC cells (right, top), (b) CAF#1 mono cultured (left, middle) and (e) co cultured with YD10B OSCC cells (right, middle) and (c) CAF#2 mono cultured(left, bottom) and (f) co cultured with YD10B OSCC cells (right, bottom) are shown at 72 h (magnification: 200X, scale bar: 2.0 mm). Enlarged images are shown in the bottom of each left.

Article Snippet: Purified plasmid DNA was transfected using 4D-nucleofector X Kit for mammalian fibroblasts and Amaxa 4D-nucleofector (Lonza, Walkersville, MD, USA), following as manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Staining

Journal: PLoS ONE

Article Title: Cytoskeletal alteration modulates cancer cell invasion through RhoA-YAP signaling in stromal fibroblasts

doi: 10.1371/journal.pone.0214553

Figure Lengend Snippet: (A)Cell size was measured on hTERT-hNOFs mono-cultured and co-cultured with HEK and OSCC cells for 3 days. ZEN 2012 software program was used (mono cultured, n = 30; with HEK, n = 23; with YD10B, n = 21; with YD38, n = 20; CAF#1, n = 22; CAF#1(with YD10B), n = 22; CAF#2, n = 16; CAF#2(with YD10B), n = 21) (* p < 0.05, ** p < 0.01, *** p < 0.001; Student t test). CAFs were used to compare cellular size in fibroblasts, respectively. Average of cellular size is shown as CAF#1, 5.74 x 10 9 nm 2 ; CAF#1 (with YD10B), 1.03 x 10 10 nm 2 ; CAF#2, 7.12 x 10 9 nm 2 and CAF#2 (with YD10B), 8.22 x 10 9 nm 2 (* p < 0.05, ** p < 0.01, *** p < 0.001; Mann Whitney U test). (B) All immunofluorescence microscopy experiments were performed on cultured cell after 3 days. DAPI(blue), Phalloidin(red) and Merged staining are shown in mono-cultured hTERT-hNOF(first panels) and hTERT-hNOF co cultured with HEK (second panels), YD10B OSCC cells (third panels) and YD38 OSCC cells (fourth panels), Scale bar, 50μm. The rectangular boxes are shown as enlarged sections to observe the F-actin assembly in left lower part.

Article Snippet: Purified plasmid DNA was transfected using 4D-nucleofector X Kit for mammalian fibroblasts and Amaxa 4D-nucleofector (Lonza, Walkersville, MD, USA), following as manufacturer’s instructions.

Techniques: Cell Culture, Software, MANN-WHITNEY, Immunofluorescence, Microscopy, Staining

Journal: PLoS ONE

Article Title: Cytoskeletal alteration modulates cancer cell invasion through RhoA-YAP signaling in stromal fibroblasts

doi: 10.1371/journal.pone.0214553

Figure Lengend Snippet: (A) Fluorescence microscope was used to check the expression of EGFP on cultured cell after transfection with pcDNA4-HisMaxB-YAP1 and YAPS127A, Scale bar, 200μm. (B) The YAP, p-YAP(S127) and αSMA protein expression was identified in all transfected cells (empty-, WT-YAP-, YAPS127A transfected hTERT-hNOFs) by Western blot. (C) Immunofluorescence images were shown in WT-YAP1- and YAPS127A-hTERT-hNOFs. Merged stained Images (DAPI(blue), F-actin(Phalloidin)(red), YAP(green))are shown. Scale bar, 50μm. The bar graph indicates the distribution of YAP in WT-YAP1- and YAPS127A-hTERT-hNOFs. It was analyzed by Image J software program (WT-YAP1-hTERT-hNOFs, n = 40; YAPS127A-hTERT-hNOFs, n = 40) (*** p < 0.001; Student t test). (D) The representative images show gel contractility by the fibroblasts. Histogram shows mean value ± SD (n = number of gels (Empty, 3; WT-YAP, 3; YAPS127A, 3), assessed over multiple experiments (* p < 0.05; Mann Whitney U test). (E) Histogram shows elastic modulus of matrix remodeled by fibroblasts, respectively. Each data point represents a single measurement, (n = 3, respectively) measurements in total. Line and error bars indicate mean value ± SD. (F-G) Representative images showed the (F) migration and (G) invasion of OSCC cells (YD10B and YD38) cultured with each of fibroblasts, which were transfected with Empty-, WT-YAP-, YAPS127A-transfected hTERT-hNOFs). Quantification of OSCC cells (YD10B and YD38) migration toward lower chamber is shown as bar graphs. Each assay was performed in independent triplicate repeats (* p < 0.05; Mann-Whitney U test).

Article Snippet: Purified plasmid DNA was transfected using 4D-nucleofector X Kit for mammalian fibroblasts and Amaxa 4D-nucleofector (Lonza, Walkersville, MD, USA), following as manufacturer’s instructions.

Techniques: Fluorescence, Microscopy, Expressing, Cell Culture, Transfection, Western Blot, Immunofluorescence, Staining, Software, MANN-WHITNEY, Migration