Additional file 1: of Serum depletion induces changes in protein expression in the trophoblast-derived cell line HTR-8/SVneo

Table S2. Preliminary assay: proteins identified via MS and bioinformatics analysis from 0.5 % serum culture. HTR-8/SVneo cells were grown in medium with 10 or 0.5 % FBS for 24 h. Total protein extracts (0.15 mg) were separated by 7 cm 2DE gels as previously described. Protein spots were analyzed via MALDI TOF and identified using the MASCOT search engine. MW (kDa) – theoretical and experimental molecular weight in kDalton; pI ~ – theoretical pI; MS score – protein score given by Mascot; % Seq – percentage sequence coverage; Pep match – number of peptides assigned to protein; Fold FBS 0.5 %/10 % – fold optical density of protein spot (expression) of 0.5 % serum proteomes (0.5 % FBS) compared to control proteomes (10 % FBS). Relevant GO terms for molecular function and biological process, retrieved by Nextprot database and DAVID tool. (XLS 57 kb

Restoration of the LKB1-AMPKα pathway in BRAF^V600E melanoma cells induces apoptosis under energy stress conditions.

<p>(A) UACC903, A375 and SKMel28 human melanoma cells were grown in complete medium (H.G.), or low glucose medium (L.G.) with or without 10 µM of the Mek1/2 specific inhibitor U0126 for 12 h. Then, Annexin V and PI (propidium iodide) positive cells were analyzed by flow cytometry. Histograms show the result from FACS analysis. Graphs on the right show the percentage of viable and dead cells in a parallel experiment under the same conditions determined by nuclear staining exclusion (Guava-ViaCount). (B) Time course at 4 and 12 hours showing the LKB1-AMPKα pathway status under the same conditions. UACC903, A375 and SKMel28 human melanoma cells were grown in complete medium (high glucose H.G.), low glucose medium (L.G.) with or without 10 µM of the Mek1/2 specific inhibitor U0126 for the times indicated. Fifty micrograms of total protein lysates were separated by SDS-PAGE and same membranes were blotted against the indicated antibodies. All experiments were done at least three times. Representative experiments are shown. (C) UACC903 cells were transfected either with a scramble siRNA or with equimolar amounts of AMPKα1 and AMPKα2 siRNAs for a total concentration of 100 nM. 72 hours after transfection cells were starved in low glucose medium for 6 hours in the presence or absence of 10 µM of U0126. Dead cells were quantified by nuclear staining exclusion (Guava-ViaCount). Western-blots show the levels of p-AMPKα^T172, AMPKα and p-Erk1/2^{Thr202/Tyr204} under the different conditions. (D) UACC903 and A375 melanoma cells were grown in complete medium (H.G. cm), serum free high glucose medium (H.G. sf), serum free low glucose medium (L.G.), serum free complete medium plus U0126 10 µM (H.G.+U0126) and low glucose serum free medium plus U0126 10 µM (L.G.+U0126) for 12 hours. The levels of Bim, phospho-Bad and Mcl-1 are showed under the different experimental conditions.</p

HGF induces LKB1^Ser431 phosphorylation in a RAS-p90^RSK dependent manner.

<p>(A) Five µg of phospho-protein isolated complexes from samples: untreated (Control), HGF triggered (40 ng/ml) w/o PHA (0,2 µM) were resolved by SDS-PAGE. p-LKB1^Ser428, p-Erk1/2^{Thr202/Tyr204} and Erk2 antibodies were probed against the membrane. Ponceau S staining of membrane is showed for phospho-protein extracts loading control. (B) 37-31E, 37-31T, SKMel28, and MeWo cells were treated in serum starvation conditions with HGF (40 ng/ml), U0126 (10 µM) and LY294002 (10 µM) as indicated in the figure. Western-blots show the levels of the indicated proteins. (C) Time course showing the phosphorylation of the LKB1^Ser431 and p-90^RSK^{Thr359/Ser363} after HGF triggering (40 ng/ml) under serum starvation conditions. LKB1 total protein is shown as a loading control. On the right, LKB1^Ser431 is phosphorylated in response to HGF in an Erk1/2-p90^RSK dependent manner. Time course shows the phosphorylation of Erk1/2^{Thr202/Tyr204} and LKB1^Ser428 in B16F1 cells. Down below, 37-31E melanoma cells were serum starved and triggered with HGF (40 ng/ml) for 5 minutes. Then, cells were treated with the Mek1/2 specific inhibitor U0126 (10 µM) for the indicated increasing times. Fifty µg of total lysates were resolved by SDS-PAGE and membrane was probed with the indicated antibodies. (D) 37-31E cells were treated for 10 min in serum starvation with HGF (40 ng/ml) in the presence or absence of U0126 (10 µM) or BI-D1870 (10 µM). Western-blots show the levels of p-LKB1^Ser431, p-Erk1/2^{Thr202/Tyr204} and p-CREB^Ser133.</p

A Comprehensive Proteome of <i>Mycoplasma genitalium</i>

<i>Mycoplasma genitalium</i> is a human pathogen associated with several sexually transmitted diseases. Proteomic technologies, along with other methods for global gene expression analysis, play a key role in understanding the mechanisms of bacterial pathogenesis and physiology. The proteome of <i>M. genitalium</i>, model of a minimal cell, has been extended using a combination of different proteomic approaches and technologies. The total proteome of this microorganism has been analyzed using gel-based and gel-free approaches, achieving the identification of 85.3% of the predicted ORFs. In addition, a comprehensive analysis of membrane subproteome has been performed. For this purpose, the TX-114 soluble fraction has been analyzed as well as the surface proteins, using cell-surface protein labeling with CyDye. Finally, the serological response of <i>M. genitalium</i>-infected patients and healthy donors has been analyzed to identify proteins that trigger immunological response. Here, we present the most extensive <i>M. genitalium</i> proteome analysis (85.3% of predicted ORFs), a comprehensive <i>M. genitalium</i> membrane analysis, and a study of the human serological response to <i>M. genitalium</i>

6569_documents_from_Agabus_454

Fasta file with the contigs of the whole transcriptome of combined specimens of Agabus ramblae (Coleoptera, Dytiscidae) after being subjected for 12h to three temperature treatments: 4ºC, control (room temperature) and 27ºC

Inhibition of oncogenic BRAF^V600E signaling restores the limited response to metabolic stress of BRAF mutant melanoma cell lines.

<p>(A) BRAF mutant melanoma cells have a limited response to energy withdrawal that is restored by U0126 treatment. Fifty micrograms of total lysates from UACC903, A375, SKMel28 and 37-31E melanoma cells grown in serum free high glucose medium (H.G.), serum free low glucose medium (L.G.) or serum free low glucose medium (L.G.) plus 10 µM of U0126 for 4 hours were separated by SDS-PAGE. Western-Blot shows the activation status of proteins in the RAS and LKB1-AMPK-mTOR pathways. (B) U0126 inhibitor treatment does not activate AMPK. UACC903 A375 and SKMel28 melanoma cells were grown in high glucose medium with serum in the absence or presence of 1 µM, 5 µM or 10 µM of U0126. Total protein lysates were subjected to SDS-PAGE. Western-blot shows the phosphorylation state of AMPKα in the presence of different concentrations of U0126. (C) Inhibition of BRAF signaling increases cell response to AICAR. UACC903 and SKMel28 cells were grown in complete medium; cells were treated with AICAR (1 mM) for 4 h in the presence or absence of U0126 (10 µM) inhibitor. p-LKB1^Ser428, p-AMPK^Thr172, p-Erk1/2^{Thr202/Tyr204}, p-ACC^Ser79 levels were checked by western blot. (D) Sorafenib treatment and siRNA BRAF knockdown restores the metabolic stress pathway in BRAF mutant melanoma cells. In the left panel, UACC903, A375 and SKMel28 melanoma cells were grown in low glucose serum free medium+/−EGF (50 ng/ml) for 4 h in the presence or absence of U0126 (10 µM) Western blots show the levels of p-AMPKα^T172, p-LKB1^Ser431 p-Erk1/2^{Thr202/Tyr204} and pCREB^Ser133 proteins under the different conditions. In the right panel SKMel28 cells were transfected with either a scramble siRNA or BRAF siRNA. 72 hours after transfection, cells were starved either in high glucose (H.G.) or low glucose (L.G.) medium for six hours. Western-blots show the levels of p-AMPKα^T172, p-Erk1/2^{Thr202/Tyr204} and BRAF proteins. (E) p90^Rsk inhibitor BI-D1870 (10 µM), does not restore the metabolic stress pathway. UACC903, A375 and SKMel28 melanoma cells were grown in low glucose serum free medium+/−EGF (50 ng/ml) for 4 h in the presence or absence of BI-D1870 (10 µM). Western blots show the levels of the indicated proteins under the different conditions.</p

TableS1

Table S1. Standardized volumes of the 563 spots common to all experiments for all treatments. Highlighted in green, spots which proteins have been identified (see Tables 2,3). RT: room temperature treatment (control). Biological replicas: r1, 2, 3

A Comprehensive Proteome of <i>Mycoplasma genitalium</i>

<i>Mycoplasma genitalium</i> is a human pathogen associated with several sexually transmitted diseases. Proteomic technologies, along with other methods for global gene expression analysis, play a key role in understanding the mechanisms of bacterial pathogenesis and physiology. The proteome of <i>M. genitalium</i>, model of a minimal cell, has been extended using a combination of different proteomic approaches and technologies. The total proteome of this microorganism has been analyzed using gel-based and gel-free approaches, achieving the identification of 85.3% of the predicted ORFs. In addition, a comprehensive analysis of membrane subproteome has been performed. For this purpose, the TX-114 soluble fraction has been analyzed as well as the surface proteins, using cell-surface protein labeling with CyDye. Finally, the serological response of <i>M. genitalium</i>-infected patients and healthy donors has been analyzed to identify proteins that trigger immunological response. Here, we present the most extensive <i>M. genitalium</i> proteome analysis (85.3% of predicted ORFs), a comprehensive <i>M. genitalium</i> membrane analysis, and a study of the human serological response to <i>M. genitalium</i>

LKB1^Ser431 (Ser428 human) is phosphorylated in response to different growth factors, in BRAF^V600E mutant melanoma cells and mouse tumor samples.

<p>(A) B16F1, 37-31T and MeWo cells were serum starved and treated with HGF (40 ng/ml), EGF (100 ng/ml), FGF2 (100 ng/ml), Herregulin (50 ng/ml), IGF-1 (50 ng/ml), PDGF (50 ng/ml), TNF-α (100 ng/ml) Insulin (100 nM) and TPA (200 nM). Fifty µg of total lysates were separated by SDS-PAGE and same membranes were incubated against the indicated antibodies. (B) MeWo (BRAF wild type), A375 (BRAF^V600E), SKMel28 (BRAF^V600E) and UACC903 (BRAF^V600E) human melanoma cells were growth in complete medium (CM) or serum starvation (SF) conditions as indicated. Fifty µg of total lysates were analyzed by SDS-PAGE. The phosphorylation status of LKB1^Ser428, p-Erk1/2^{Thr202/Tyr204} and p-90^{RSK Thr359/Ser363} is shown. Total Erk1/2 is used as a loading control. Cell genotypes are showed. (C) p-LKB1^Ser431 and p-Erk1/2^{Thr202/Tyr204} levels in mouse melanoma tumor samples. Samples 1–7 primary tumors raised in HGF-UV irradiated transgenic mice. Samples 8 and 9 show xenograph tumors generated from 37-31E cells in FVB mice with high and low p-Erk1/2 levels, respectively. As a control fifty micrograms of protein from 37-31E melanoma cell line treated with HGF (40 ng/ml) for 10 minutes was added (Total lysates, T.L.). Same membrane was blotted against the indicated antibodies. Quantifications of phospho-proteins normalized against total protein are showed in the graphs below.</p

A Comprehensive Proteome of <i>Mycoplasma genitalium</i>

<i>Mycoplasma genitalium</i> is a human pathogen associated with several sexually transmitted diseases. Proteomic technologies, along with other methods for global gene expression analysis, play a key role in understanding the mechanisms of bacterial pathogenesis and physiology. The proteome of <i>M. genitalium</i>, model of a minimal cell, has been extended using a combination of different proteomic approaches and technologies. The total proteome of this microorganism has been analyzed using gel-based and gel-free approaches, achieving the identification of 85.3% of the predicted ORFs. In addition, a comprehensive analysis of membrane subproteome has been performed. For this purpose, the TX-114 soluble fraction has been analyzed as well as the surface proteins, using cell-surface protein labeling with CyDye. Finally, the serological response of <i>M. genitalium</i>-infected patients and healthy donors has been analyzed to identify proteins that trigger immunological response. Here, we present the most extensive <i>M. genitalium</i> proteome analysis (85.3% of predicted ORFs), a comprehensive <i>M. genitalium</i> membrane analysis, and a study of the human serological response to <i>M. genitalium</i>