Journal: Cells

Article Title: Atypical Exon 2/3 Mutants G48C, Q43K, and E37K Present Oncogenic Phenotypes Distinct from Characterized NRAS Variants

doi: 10.3390/cells13201691

Figure Lengend Snippet: Effect of the novel NRAS mutants on cell proliferation. NRAS G48C, Q43K, and E37K enhanced proliferation of both HCT116 and NIH3T3 cells compared to vector-only, wild-type, and canonical mutant control Q61K. Proliferation rates of ( a ) HCT116 cells and ( b ) NIH3T3 cells transfected with empty vector, wild-type NRAS, canonical NRAS Q61K mutant, and the novel NRAS mutants G48C, Q43K, and E37K are shown. Data presented are representative of three independent trials, each performed in triplicate, and expressed as mean ± standard deviation (SD). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. PTT = pTargeT vector-only control; WT = wild-type; ns = non-significant.

Article Snippet: Two cell lines were used in this study, as follows: the NIH3T3 murine embryonic fibroblast cell line (Cat. No. CCL-247) and the HCT116 human colorectal carcinoma cell line (Cat. No. CCL-247), which were both obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Plasmid Preparation, Mutagenesis, Control, Transfection, Standard Deviation

Journal: Cells

Article Title: Atypical Exon 2/3 Mutants G48C, Q43K, and E37K Present Oncogenic Phenotypes Distinct from Characterized NRAS Variants

doi: 10.3390/cells13201691

Figure Lengend Snippet: Effect of the novel NRAS mutants, G48C, Q43K, and E37K, on cell survival. ( a ) HCT116 and ( b ) NIH3T3 cells were transfected with NRAS WT and mutant constructs, then assessed for their ability to resist apoptosis when induced with sodium butyrate (HCT116) and reduced serum concentration (NIH3T3). Data presented are representative of three independent trials, each performed in triplicate, and expressed as mean ± standard deviation (SD). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. PTT = pTargeT vector-only control; WT = wild-type; ns = non-significant.

Article Snippet: Two cell lines were used in this study, as follows: the NIH3T3 murine embryonic fibroblast cell line (Cat. No. CCL-247) and the HCT116 human colorectal carcinoma cell line (Cat. No. CCL-247), which were both obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Transfection, Mutagenesis, Construct, Concentration Assay, Standard Deviation, Plasmid Preparation, Control

Journal: Cells

Article Title: Atypical Exon 2/3 Mutants G48C, Q43K, and E37K Present Oncogenic Phenotypes Distinct from Characterized NRAS Variants

doi: 10.3390/cells13201691

Figure Lengend Snippet: Effect of NRAS G48C, Q43K, and E37K mutants on cellular migration in HCT116 and NIH3T3 cells. Scratch wound assay showing representative images of ( a ) HCT116 and ( c ) NIH3T3 cells transfected with empty vector, wild-type NRAS, canonical NRAS Q61K control, and the novel NRAS mutants G48C, Q43K, and E37K at 0 h and 16 h post-scratch. The migration rates for each setup are shown in ( b , d ) for HCT116 and NIH3T3 cells, respectively. Data presented are representative of three independent trials, each performed in triplicate, and expressed as mean ± standard deviation (SD). ** p ≤ 0.01. pTT = pTargeT vector-only control; WT = wild-type; ns = non-significant.

Article Snippet: Two cell lines were used in this study, as follows: the NIH3T3 murine embryonic fibroblast cell line (Cat. No. CCL-247) and the HCT116 human colorectal carcinoma cell line (Cat. No. CCL-247), which were both obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Scratch Wound Assay Assay, Transfection, Plasmid Preparation, Control, Standard Deviation

Journal: Cells

Article Title: Atypical Exon 2/3 Mutants G48C, Q43K, and E37K Present Oncogenic Phenotypes Distinct from Characterized NRAS Variants

doi: 10.3390/cells13201691

Figure Lengend Snippet: Expression of NRAS mutants induce cytoskeletal remodeling in NIH3T3 cells. Distinct features, such as prominent peripheral stress fibers (yellow arrows) and highly organized actin filaments (red arrowheads), are present in both the empty vector ( A ) and wild-type ( B ) setups. Central round nuclei (orange arrowheads) are also visible in WT. Changes in cytoskeletal architecture, as well as invasive structures, are observable in both canonical and novel mutant setups ( C , D = Q61K; E , F = G48C; G , H = Q43K; I , J = E37K), including fan-shaped cells with prominent migrating front (sky blue brackets); peripheral dorsal ruffles (pink arrowheads); circular dorsal ruffles (yellow arrowheads); tunneling nanotubes (gray arrowheads); multilobulated nuclei (red arrows); invadopodia (pink arrows); lamellipodia (gold arrowheads); filopodia (purple arrowheads); and fusing cells (yellow brackets).

Article Snippet: Two cell lines were used in this study, as follows: the NIH3T3 murine embryonic fibroblast cell line (Cat. No. CCL-247) and the HCT116 human colorectal carcinoma cell line (Cat. No. CCL-247), which were both obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Plasmid Preparation, Mutagenesis

Journal: Biomedical Optics Express

Article Title: Method for nanoparticles uptake evaluation based on double labeled fluorescent cells scanned in enhanced darkfield microscopy

doi: 10.1364/BOE.490136

Figure Lengend Snippet: a) Fluorescence image of a group of NIH3T3 cells (experimental colors through F4 filter); b) cross-section from F1-Z-stack; c) cross-section from F2-Z-stack; d) deconvolved image of b; e) deconvolved image of c; f) cross-section from WL-Z-stack with localized ZnO-NPs; g)-i) movie frames obtained by combining all Z-stacks (digital colors).

Article Snippet: Murine embryonic fibroblast cell line NIH3T3 (ATCC-CRL 1658) was used.

Techniques: Fluorescence

Journal: Biomedical Optics Express

Article Title: Method for nanoparticles uptake evaluation based on double labeled fluorescent cells scanned in enhanced darkfield microscopy

doi: 10.1364/BOE.490136

Figure Lengend Snippet: The metabolic viability of NIH3T3 cells incubated for 24 h with different concentrations of ZnO-NPs.

Article Snippet: Murine embryonic fibroblast cell line NIH3T3 (ATCC-CRL 1658) was used.

Techniques: Incubation

Journal: PLoS ONE

Article Title: RECK-Mediated β1-Integrin Regulation by TGF-β1 Is Critical for Wound Contraction in Mice

doi: 10.1371/journal.pone.0135005

Figure Lengend Snippet: (A) NIH3T3 fibroblasts and were incubated with TGF-β1 for 24 and 48 h at the indicated concentrations. Western blots analysis of cell extracts were performed to determine the expression of RECK, β1-integrin, FN and CTGF. β-tubulin (Tubulin) levels were used as a loading control. (B) Primary fibroblasts cultures derived from tibialis anterior skeletal muscles (SM) and skin biopsies of 3-month-old WT mice were incubated with TGF-β1 for 24 as indicated. Western blot analysis of cell extracts were performed to determine the levels of RECK, FN, and CTGF. tubulin levels were used as a loading control. (C) NIH3T3 fibroblasts were incubated with 5 ng/ml of TGF-β1 for the indicated times. At the end of the assay, total RNA was extracted and was reverse transcribed into complementary DNA. Taqman quantitative real-time PCR was performed to determine Reck expression. mRNA expression was quantified using the comparative ΔC T method (2 -ΔΔCT ) using Gadph as a reference gene. mRNA levels are presented relative to the mean expression of the control (untreated cells). (D) Reck mRNA expression in NIH3T3 fibroblasts incubated with different concentrations of TGF-β1 for 24 hours was determined as in (C). In C and D, values are expressed as mean +/- standard deviation (SD) of two independent experiments. In C, &, P<0.05 relative to 0 hour; #, P<0.05 relative to 6 hours. In D, &, P<0.05 relative to 0 ng/ml; #, P<0.05 relative to 0,5 ng/ml.

Article Snippet: The murine embryonic fibroblasts cell lines NIH3T3 were obtained from American Type Culture Collection (ATCC) (Rockville, MD, USA) and grown as previously described [ ].

Techniques: Incubation, Western Blot, Expressing, Control, Derivative Assay, Muscles, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: PLoS ONE

Article Title: RECK-Mediated β1-Integrin Regulation by TGF-β1 Is Critical for Wound Contraction in Mice

doi: 10.1371/journal.pone.0135005

Figure Lengend Snippet: (A) NIH3T3 fibroblasts were pre-treated for 30 minutes with different inhibitors: TGF-β-RI kinase inhibitor SB525334, PI3K inhibitor LY294002, MEK1 inhibitor PD98059, JNK inhibitor SB600125, and p38 inhibitor SB203580. After the pre-treatment, fibroblasts were treated with 5ng/ml of TGF-β1 for 24 hours or untreated to serve as controls. Western blot analysis of cell extracts were performed to determine the levels of RECK and FN. tubulin levels were used as a loading control. (B) NIH3T3 fibroblasts were transiently transfected with a pool of siRNA against Smad-2 and Smad-3 or with a scrambled siRNA as a control. 24 h post-transfection, cells were treated with 5 ng/ml of TGF-β1 for 24 h, or left untreated to serve as a control. Western blot analysis of cell extracts were performed to determine the levels of RECK, Smad-3 and β1-Integrin. β-tubulin levels were used as a loading control. (C) NIH3T3 fibroblasts were pre-treated for 30 minutes with SIS3, a specific inhibitor of Smad-3 activation, and the JNK inhibitor SB600125; cells were treated either alone or in combination. Afterwards, fibroblasts were treated with 5 ng/ml of TGF-β1, or left untreated as a control, for 24h. Western blot analysis of cell extracts were performed to determine the levels of RECK and β1-Integrin. β-tubulin levels were used as a loading control. The quantifications shown in A, B and C are from two independent. Statistical significance was assessed using two-way ANOVA and a Bonferroni multiple-comparison post hoc test. &, P<0.05 relative to TGF-β1 untreated fibroblasts.

Article Snippet: The murine embryonic fibroblasts cell lines NIH3T3 were obtained from American Type Culture Collection (ATCC) (Rockville, MD, USA) and grown as previously described [ ].

Techniques: Western Blot, Control, Transfection, Activation Assay, Comparison

Journal: PLoS ONE

Article Title: RECK-Mediated β1-Integrin Regulation by TGF-β1 Is Critical for Wound Contraction in Mice

doi: 10.1371/journal.pone.0135005

Figure Lengend Snippet: (A) NIH3T3 fibroblasts were transiently transfected with a pool of shRNAs against mouse RECK (shRECK) or with a scrambled sequence as control (shCtrl). 24 h post-transfection, cells were treated or not with 5 ng/ml of TGF-β1 for 24 h ( B ) NIH3T3 fibroblasts were transiently transfected with a vector that overexpresses RECK or with an empty vector as control. 24 h post-transfection, cells were treated with 5 ng/ml of TGF-β1 for 24 h, or left untreated as a control. In A and B, Western blot analysis of cell extracts were performed to determine the levels of RECK, β1-integrin and CTGF. Tubulin levels were used as a loading control. In A, &, P<0.05 relative to untreated shCtrl; #, P<0.05 relative to untreated shRECK. In B, &, P<0.05 relative to TGF-β1 untreated Empty vector; #, P<0.05 relative to TGF-β1 untreated RECK; ¢, P<0.05 relative to TGF-β1 treated Empty vector.

Article Snippet: The murine embryonic fibroblasts cell lines NIH3T3 were obtained from American Type Culture Collection (ATCC) (Rockville, MD, USA) and grown as previously described [ ].

Techniques: Transfection, Sequencing, Control, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: RECK-Mediated β1-Integrin Regulation by TGF-β1 Is Critical for Wound Contraction in Mice

doi: 10.1371/journal.pone.0135005

Figure Lengend Snippet: (A) Schematic of the matrix contraction assay. From top to bottom, fibroblasts were mixed with a collagen solution that polymerizes into a 3D collagen matrix for 2 h. This mixture was then released from the culture dish and allowed to float in culture medium. Floating collagen lattices were incubated with TGF-β1 for 24 hours to induce fibroblasts contraction, leading to a volume reduction of the collagen lattice. (B) NIH3T3 fibroblasts were transiently transfected with a pool of shRNAs against mouse RECK (shReck) or with a RECK overexpression vector or with an empty vector as control. 24 h post-transfection, fibroblasts were trypsinized and a collagen contraction assay was performed as described in (A). After 24 h, the now trypsinized collagen lattices were treated with TGF-β1 and/or the focal adhesion kinase inhibitor 14 (FAK-Inh) for 24 h. Afterwards, 3D floating matrices were photographed. Representative images are shown. (C) The volume of the contracted matrices obtained in (B) was measured immediately after being released at the end of the assay. These values, obtained from two independent experiments performed in triplicate, were graphed as a percentage of the initial volume. Statistical significance was assessed using two-way ANOVA and a Bonferroni multiple-comparison post hoc test. &, P<0.05 relative to TGF-β1 untreated Empty vector; #, P<0.05 relative to TGF-β1 untreated RECK; ¢, P<0.05 relative to TGF-β1 treated Empty vector; ∞, P<0.05 relative to TGF-β1 treated RECK. (D) Aliquots of the trypsinized cells in (B) were directly seeded in culture dishes before mixing with the collagen solution and then treated with TGF-β1 and/or the FAK-Inh for 24 has described in (B). Cell extracts were prepared and analyzed by Western blot to determine the levels of RECK and β1-integrin. Tubulin levels were used as a loading control.

Article Snippet: The murine embryonic fibroblasts cell lines NIH3T3 were obtained from American Type Culture Collection (ATCC) (Rockville, MD, USA) and grown as previously described [ ].

Techniques: Contraction Assay, Incubation, Transfection, Over Expression, Plasmid Preparation, Control, Comparison, Western Blot