Journal: PLoS Biology

Article Title: The Rho Exchange Factors Vav2 and Vav3 Favor Skin Tumor Initiation and Promotion by Engaging Extracellular Signaling Loops

doi: 10.1371/journal.pbio.1001615

Figure Lengend Snippet: (A) Scheme of these experiments. (B, C) Example (B; scale bar, 100 µm) and quantification (C) of apoptotic cells found in the epidermis of mice of the indicated genotypes 24 h upon the application of DMBA ( n = 4). In (B), sections were stained with antibodies to the cleaved fragment of caspase 3 (ID number: 12367) and 14, 4′,6-diamidino-2-phenylindole (DAPI) to reveal apoptotic cells (red color) and cell nuclei (blue color), respectively. (D, E) Example (D; scale bar, 100 µm) and quantification (E) of phospho-histone H2AX + keratinocytes (brown color) present in the epidermis of short-term DMBA-treated mice of indicated genotypes ( n = 4). p-, phosphorylated. (F) Number of apoptotic (annexin V + ) wild-type and Vav2 −/− ; Vav3 −/− keratinocytes induced after 8 (left panel) and 12 (right panel) h in the indicated culture conditions ( n = 4). Bleo, bleomycin; DTT, dithiothreitol; NS, no statistically significant. (G, H) Example of a flow cytometry experiment (G) and subsequent quantification (H) of the level of apoptosis induced by either DMBA (G, H) or serum starvation (H) in wild-type and Vav2 −/− ; Vav3 −/− keratinocytes ectopically expressing GFP either alone (G, H) or in combination with HA-Vav2 (H) or Myc-Vav3 (G, H) ( n = 3). In (G), only the gated GFP + cells are shown.

Article Snippet: In vitro cell cycle transitions were determined using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen).

Techniques: Staining, Flow Cytometry, Expressing

Journal: PLoS Biology

Article Title: The Rho Exchange Factors Vav2 and Vav3 Favor Skin Tumor Initiation and Promotion by Engaging Extracellular Signaling Loops

doi: 10.1371/journal.pbio.1001615

Figure Lengend Snippet: (A) Quantification by flow cytometry of the number of EdU + , S-phase cells induced by the stimulation of quiescent keratinocytes of the indicated genotypes with either TPA or a synthetic keratinocyte growth media (CnT07). (B) Phosphorylation and expression status of Erk and Stat3 proteins in TPA-stimulated keratinocytes of indicated genotypes. (C) Rac1 (upper panel) and RhoA (lower panel) activation levels induced by the stimulation of quiescent keratinocytes of indicated genotypes with TPA ( n = 3). (D) Phosphorylation and expression status of Erk proteins in either serum- (two upper panels) or CnT07-stimulated (two bottom panels) keratinocytes of indicated genotypes. (E) Rac1 activation levels induced by the stimulation of quiescent keratinocytes with either serum (left panel) or CnT07 media (right panel) ( n = 3). (F) Immunoprecipitation experiments showing the tyrosine phosphorylation levels of endogenous Vav2 (top panel) and ectopically expressed HA-Vav2 (third panel from top) in wild-type keratinocytes treated with TPA (+) in the absence (−) or the presence (+) of the indicated drugs ( n = 2). Due to problems with the detection of the endogenous proteins after blot stripping, the loading control shown in the second panel from top was made using a parallel immunoprecipitation with the same anti-Vav2 antibody. GF, GF109203X; IP, immunoprecipitation. (G) Western blot of total cellular extracts showing the levels of phosphorylation (top) and expression (bottom) of Erk proteins in wild-type keratinocytes stimulated as indicated in (F) ( n = 3). (H) Rac1 activation levels induced by TPA in wild-type keratinocytes under the indicated experimental conditions ( n = 3). Go, Gö 6976; sh Fyn , cells infected with a Fyn-specific shRNA.

Article Snippet: In vitro cell cycle transitions were determined using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen).

Techniques: Flow Cytometry, Expressing, Activation Assay, Immunoprecipitation, Stripping, Western Blot, Infection, shRNA

Journal: PLoS Biology

Article Title: The Rho Exchange Factors Vav2 and Vav3 Favor Skin Tumor Initiation and Promotion by Engaging Extracellular Signaling Loops

doi: 10.1371/journal.pbio.1001615

Figure Lengend Snippet: (A) Quantification by flow cytometry of the apoptosis induced in pure (No) and genotypically mixed (Yes) cultures of wild-type and Vav2 −/− ; Vav3 −/− keratinocytes upon 12 h in complete media with DMBA or under serum-free media conditions. (B) Quantification by flow cytometry of the apoptosis observed in wild-type and Vav2 −/− ; Vav3 −/− keratinocytes kept for 12 h with serum, without serum, or without serum plus the indicated extracellular factors ( n = 3). (C) Determination by flow cytometry of the percentage of wild-type and Vav2 −/− ; Vav3 −/− keratinocytes that have entered S phase after 4.5 h under the indicated culture conditions ( n = 3). (D–F) Phosphorylation and expression status of Erk and Stat3 in serum-starved wild-type and Vav2 −/− ; Vav3 −/− keratinocytes stimulated with either EGF (D) or IL6 (E–G) for the indicated periods of time. In panels (F) and (G), keratinocytes were transduced with empty and Vav-encoding lentiviruses prior to the starvation and stimulation steps as indicated in the respective panel ( n = 3). (H) Tyrosine phosphorylation levels of endogenous Vav2 (top panel) in wild-type keratinocytes treated with the indicated agents for 5 min ( n = 2). Due to problems with the detection of the endogenous proteins after blot stripping, the loading control shown in the second panel from top was made using a parallel immunoprecipitation with the same anti-Vav2 antibody.

Article Snippet: In vitro cell cycle transitions were determined using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen).

Techniques: Flow Cytometry, Expressing, Transduction, Stripping, Immunoprecipitation