Journal:

Article Title: CDC42 and FGD1 Cause Distinct Signaling and Transforming Activities

doi:

Figure Lengend Snippet: CDC42(12V) and FGD1 cooperate with Raf-1 in NIH 3T3 focus formation assays. (a) NIH 3T3 cells were cotransfected with pZIP-NeoSV(x)1 or pAX142 expression plasmids encoding the indicated proteins. Five hundred nanograms of each construct was transfected per 60-mm-diameter dish. Foci of transformed cells were counted at 14 days. Data shown are from one experiment performed in duplicate and are representative of three independent experiments. Error bars, standard errors. (b) FGD1 and CDC42(12V) cooperate with Raf(340D) to cause transformed foci with distinct morphologic appearances. Foci were photographed under phase microscopy at 14 days.

Article Snippet: Expression was determined by Western blot analysis using an anti-Myc antibody (9E10; Santa Cruz Biotech). (b) NIH 3T3 cells that constitutively express FGD1 cause Rho-like transformed foci in a secondary focus formation assay.

Techniques: Expressing, Construct, Transfection, Transformation Assay, Microscopy

Journal:

Article Title: CDC42 and FGD1 Cause Distinct Signaling and Transforming Activities

doi:

Figure Lengend Snippet: FGD1 causes growth transformation of NIH 3T3 cells. NIH 3T3 cells were transfected with 3 μg of pRK5 or pRK5-myc-FGD1 and then selected for 14 days with G418-containing growth medium (200 μg/ml). (a) Expression of FGD1 and FGD1Δ. The upper panel shows FGD1 expression in a stably selected NIH 3T3 cell line. The lower panel shows transient expression of FGD1 and FGD1Δ in 293 cells. Expression was determined by Western blot analysis using an anti-Myc antibody (9E10; Santa Cruz Biotech). (b) NIH 3T3 cells that constitutively express FGD1 cause Rho-like transformed foci in a secondary focus formation assay. Stably selected cells (103) were mixed with 106 untransformed cells and then plated. Foci were counted and photographed at 7 days. (c) FGD1 promotes anchorage-independent growth in soft agar when constitutively expressed in NIH 3T3 cells. The FGD1-transfected cell line was seeded at 5 × 104 cells per 60-mm-diameter dish in growth medium containing 0.3% agar over a base layer of 0.6%. Colonies were counted and photographed at 21 days.

Article Snippet: Expression was determined by Western blot analysis using an anti-Myc antibody (9E10; Santa Cruz Biotech). (b) NIH 3T3 cells that constitutively express FGD1 cause Rho-like transformed foci in a secondary focus formation assay.

Techniques: Transformation Assay, Transfection, Expressing, Stable Transfection, Western Blot, Tube Formation Assay

Journal:

Article Title: CDC42 and FGD1 Cause Distinct Signaling and Transforming Activities

doi:

Figure Lengend Snippet: FGD1 causes tumorigenic transformation of NIH 3T3 cells

Article Snippet: Expression was determined by Western blot analysis using an anti-Myc antibody (9E10; Santa Cruz Biotech). (b) NIH 3T3 cells that constitutively express FGD1 cause Rho-like transformed foci in a secondary focus formation assay.

Techniques: Transformation Assay, Activity Assay, Plasmid Preparation

Journal:

Article Title: CDC42 and FGD1 Cause Distinct Signaling and Transforming Activities

doi:

Figure Lengend Snippet: FGD1 and CDC42 stimulate transcription from common promoter elements. NIH 3T3 cells were transfected with 1.5 μg of pAX142, pAX142-myc-FGD1, pAX142-myc-FGD1Δ, or pAX142-cdc42(12V) along with luciferase gene reporter constructs for SRF transcriptional activity (2.5 μg) (A), c-Jun transcriptional activity (500 ng of Gal-Jun and 2.5 μg 5×Gal) (B), Elk-1 transcriptional activity (500 ng of Gal-Elk and 2.5 μg of 5×Gal) (C), or cyclin-D1 transcription (2.5 μg of CD1-luciferase) (D). Following transfection, cells were cultured for 30 h and then serum starved (0.5% calf serum) for 14 h before extract preparation. Luciferase activity was determined and expressed as fold activation relative to the level of activation seen with the vector controls. Data shown are representative of three independent experiments performed on duplicate plates. Error bars, standard errors.

Article Snippet: Expression was determined by Western blot analysis using an anti-Myc antibody (9E10; Santa Cruz Biotech). (b) NIH 3T3 cells that constitutively express FGD1 cause Rho-like transformed foci in a secondary focus formation assay.

Techniques: Transfection, Luciferase, Construct, Activity Assay, Cell Culture, Activation Assay, Plasmid Preparation

Journal:

Article Title: CDC42 and FGD1 Cause Distinct Signaling and Transforming Activities

doi:

Figure Lengend Snippet: FGD1 and CDC42(12V) cooperate with Raf to stimulate Elk-1 but not SRF or c-Jun transcriptional activity. NIH 3T3 cells were cotransfected with pAX142 or pZIP-NeoSV(x)1 (500 ng) expression plasmids encoding the indicated proteins along with luciferase gene reporter constructs for SRF transcriptional activity (2.5 μg) (A), c-Jun transcriptional activity (500 ng of Gal-Jun and 2.5 μg of 5×Gal) (B), or Elk-1 transcriptional activity (500 ng of Gal-Elk and 2.5 μg of 5×Gal) (C). Transcriptional assays were performed as described in the legend to Fig. ​Fig.3.3. Data shown are representative of three independent experiments performed on duplicate plates. Error bars, standard errors.

Article Snippet: Expression was determined by Western blot analysis using an anti-Myc antibody (9E10; Santa Cruz Biotech). (b) NIH 3T3 cells that constitutively express FGD1 cause Rho-like transformed foci in a secondary focus formation assay.

Techniques: Activity Assay, Expressing, Luciferase, Construct

Journal:

Article Title: CDC42 and FGD1 Cause Distinct Signaling and Transforming Activities

doi:

Figure Lengend Snippet: FGD1 stimulates SRF, Elk-1, and c-Jun activity in a CDC42-dependent manner. NIH 3T3 cells were cotransfected with expression plasmids encoding the indicated proteins (1.5 μg of pAX142, pAX142-myc-FGD1, pAX142-cdc42(12V), or pAX142-cdc42(17N); 0.25 μg of pyDF30 or pyDF30-WASP-GBD) along with luciferase gene reporter constructs for SRF transcriptional activity (2.5 μg) (A), c-Jun transcriptional activity (500 ng of Gal-Jun and 2.5 μg of 5×Gal) (B), or Elk-1 transcriptional activity (500 ng of Gal-Elk and 2.5 μg of 5×Gal) (C). Transcriptional assays were performed as described in the legend to Fig. ​Fig.3.3. Data shown are representative of three independent experiments performed on duplicate plates. Error bars, standard error.

Article Snippet: Expression was determined by Western blot analysis using an anti-Myc antibody (9E10; Santa Cruz Biotech). (b) NIH 3T3 cells that constitutively express FGD1 cause Rho-like transformed foci in a secondary focus formation assay.

Techniques: Activity Assay, Expressing, Luciferase, Construct

Journal:

Article Title: CDC42 and FGD1 Cause Distinct Signaling and Transforming Activities

doi:

Figure Lengend Snippet: FGD1 cooperates with RhoA and CDC42 but not Rac1 to stimulate transcriptional response elements. NIH 3T3 cells were cotransfected with pAX142 expression plasmids encoding the indicated proteins (0.5 μg of pAX142, pAX142-cdc42, pAX142-FGD1, pAX142-lsc-D7HA, and pAX142-Dbl-HA1 [a] or 0.5 μg of pAX142, pAX142-FGD1, pAX142-rhoA, pAX142-rac1, or pAX142-cdc42 [b]) along with luciferase gene reporter constructs for SRF transcriptional activity (2.5 μg) (A), c-Jun transcriptional activity (500 ng of Gal-Jun and 2.5 μg of 5×Gal) (B), or Elk-1 transcriptional activity (500 ng of Gal-Elk and 2.5 μg of 5×Gal) (C). Transcriptional assays were performed as described in the legend to Fig. ​Fig.3.3. Data shown are representative of three independent experiments performed on duplicate plates. Error bars, standard errors.

Article Snippet: Expression was determined by Western blot analysis using an anti-Myc antibody (9E10; Santa Cruz Biotech). (b) NIH 3T3 cells that constitutively express FGD1 cause Rho-like transformed foci in a secondary focus formation assay.

Techniques: Expressing, Luciferase, Construct, Activity Assay

Journal:

Article Title: CDC42 and FGD1 Cause Distinct Signaling and Transforming Activities

doi:

Figure Lengend Snippet: FGD1 stimulates SRF and Elk-1 but not c-Jun activity in a RhoA-dependent manner. NIH 3T3 cells were cotransfected with expression plasmids encoding the indicated proteins [1.5 μg of pAX142, pAX142-myc-FGD1, and pAX142-rhoA(19N)] along with luciferase gene reporter constructs for SRF transcriptional activity (2.5 μg) (A), c-Jun transcriptional activity (500 ng of Gal-Jun and 2.5 μg of 5×Gal) (B), or Elk-1 transcriptional activity (500 ng of Gal-Elk and 2.5 μg of 5×Gal) (C). Transcriptional assays were performed as described in the legend to Fig. ​Fig.3.3. Data shown are representative of three independent experiments performed on duplicate plates. Error bars, standard errors.

Article Snippet: Expression was determined by Western blot analysis using an anti-Myc antibody (9E10; Santa Cruz Biotech). (b) NIH 3T3 cells that constitutively express FGD1 cause Rho-like transformed foci in a secondary focus formation assay.

Techniques: Activity Assay, Expressing, Luciferase, Construct

Journal:

Article Title: An Elaborate Pathway Required for Ras-Mediated Epigenetic Silencing

doi: 10.1038/nature06251

Figure Lengend Snippet: a, Schematic summary of the genome-wide shRNA screen for Ras-mediated epigenetic silencing of Fas. b, Immunoblot analysis monitoring Fas expression in the 28 K-ras NIH 3T3 knockdown (KD) cell lines. Expression of Fas in K-ras NIH 3T3 cells in the presence and absence of 5-aza-2’-deoxycytidine (5-aza) is also shown. K-Ras expression is shown as a loading control.

Article Snippet: 5×10 6 NIH 3T3, K- ras NIH 3T3, or K- ras NIH 3T3 knockdown Cells were injected subcutaneously into the right flank of athymic Balb/c (nu/nu) mice (Taconic).

Techniques: Genome Wide, shRNA, Western Blot, Expressing, Knockdown, Control

Journal:

Article Title: An Elaborate Pathway Required for Ras-Mediated Epigenetic Silencing

doi: 10.1038/nature06251

Figure Lengend Snippet: a, qRT-PCR monitoring expression of Fas, Sfrp1, Par4, Plagl1, H2-K1 and Lox in NIH 3T3 cells, and in K-ras NIH 3T3 cells in the presence and absence of 5-aza. Values are expressed as fold re-expression relative to expression of the gene in K-ras NIH 3T3 cells, which is arbitrarily set to 1. Error bars indicate standard error (n=3). b, Summary of qRT-PCR analysis monitoring re-expression of Fas, Sfrp1, Par4, Plagl1, H2-K1 and Lox following knockdown of each of the 28 RESEs.

Article Snippet: 5×10 6 NIH 3T3, K- ras NIH 3T3, or K- ras NIH 3T3 knockdown Cells were injected subcutaneously into the right flank of athymic Balb/c (nu/nu) mice (Taconic).

Techniques: Quantitative RT-PCR, Expressing, Knockdown