multiG2

M. DUTILLEUL, J.M. BONZOM, C. LECOMTE, B. GOUSSEN, F. DAIAN, S. GALAS, D. REALE 2014 Rapid evolutionary responses of life history traits to different experimentally-induced pollution in Caenorhabditis elegans BMC Evolutionary Biology =============================== File name: multiG2.csv File description: measures of survival, sex ratio and population size of Caenorhabditis elegans populations in different environments during a multi-generationnal experiment. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Definition of column headings: generation: generation in which population was measured (1 to 22) environment: environment where the population lived (control, salt, uranium, alternating) replicate: denomination of the replicate population through the generations (A to X) survival: percentage of survival in the replicate population (%) ratio_M: percentage of male in the replicate population (%) pop_size: number of individuals in the replicate population Missing data are indicated with NA ================================ Contact information: [email protected]

multiG1

M. DUTILLEUL, J.M. BONZOM, C. LECOMTE, B. GOUSSEN, F. DAIAN, S. GALAS, D. REALE 2014 Rapid evolutionary responses of life history traits to different experimentally-induced pollution in Caenorhabditis elegans BMC Evolutionary Biology ================================= File name: multiG1.csv File description: measures of life history traits of Caenorhabditis elegans in different environments during a multi-generationnal experiment. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Definition of column headings: generation: generation in which the individual* was measured (1 to 22)* environment: environment where the individual* lived (control, salt, uranium, alternating) replicate: population from where the individual* was sampled (A to X)* fertility_B96: number of larvae produced by the hermaphrodite before 96h of life fertility_A96: number of larvae produced by the hermaphrodite after 96h of life fertility_tot: number of larvae produced by the hermaphrodite in its all life length_H: body length of the hermaphrodite (in mm) bend_M: number of body bend of the male in 20 seconds length_M: body length of the male (in mm). Missing data are indicated with NA *Although the data are on the same line to shorten the table, the hermaphrodite and the male measured at each line came from a same replicate but were obviously different individuals =============================== Contact information: [email protected]

Triflorcas blocks Met-triggered cell survival and in vitro tumorigenesis of human NSCLC cells carrying Met mutations (H2212 and H1437) and of human gastric carcinoma cells carrying Met amplification (GTL-16).

<p>(A) Survival of H2122 and GTL-16 cells was reduced by Triflorcas at indicated concentration (µM; n = 3). In contrast, SU11274 (SU), crizotinib (crizo), and PHA665752 (PHA) impaired survival of GTL-16, but not of H2212 cells. Cells were serum-starved for 24 hours and then incubated with compounds for 48 hours. (B) Triflorcas blocked anchorage-independent growth of H1437 and GTL-16 cells in a dose dependent manner (n = 3). SU11274, crizotinib, and PHA665752 impaired in vitro tumorigenesis of GTL-16, but not of H1437 cells. Values are expressed as means ± s.e.m. **P<0.01; ***P<0.001; Student-<i>t</i> test.</p

Small molecule kinase interaction map for Triflorcas.

<p>Compound was screened against a KINOME<i>scan</i> (<a href="http://www.kinomescan.com" target="_blank">http://www.kinomescan.com</a>) panel of 98 kinases. (A) The table indicates the kinases for which Triflorcas reduced more than 30% the binding constant. The ligand binding for each kinase (% of control condition) is indicated. (B) TREEspot image of the 98 kinases screened with the position of Triflorcas targets: Abl wild-type, Abl E255K and Abl T315I mutant forms, IKK-beta, JAK2, MKNK1, and ZAP70. The complete dataset is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046738#pone.0046738.s008" target="_blank">Table S2</a>. TK: tyrosine kinase; TKL: tyrosine kinase like; STE: STE kinase; CK1: cell kinase1; AGC: PKA, PKC, PKG kinases; CAMK: calcium calmodulin-regulated kinase; CMGC: CDK, MAP, GSK, CDK-like kinase.</p

Triflorcas perturbs cell cycle progression, leading to mitotic failure.

<p>(A) Graph showing the ratio of percentage of GTL-16 cells in G0/G1 phase over the percentage of cells in S+G2/M phase. Cells were treated with Triflorcas (TFC; 3 µM), or vehicle (cntr) for 48 hours, then stained with propidium iodide. Values are expressed as means ± s.e.m. ***P<0.001; Student-<i>t</i> test (n = 3). Percentages of cells in G0/G1, S, and G2/M phases are reported in the table. (B) GTL-16 cells were grown in the presence of Triflorcas or vehicle and nuclei were visualized using DAPI staining. Arrows show example of cells with multiple nuclei (40X magnification). (C) Quantification of cells with mitotic failure expressed as percentage of the total number of cells analyzed. Triflorcas (TFC; 3 or 10 µM), but not SU11274 (SU; 1 µM), crizotinib (crizo; 1 µM), or PHA665752 (PHA; 1 µM) led to a significant increase in the number of cells with mitotic failure. Values are expressed as means ± s.e.m. ***P<0.001; Student-<i>t</i> test.</p

Triflorcas impairs in vivo tumor growth of cancer cells carrying oncogenic Met, without causing major side effects.

<p>(A) Evolution of the body weight in mice, treated intra-peritoneally with either vehicle or Triflorcas (TFC; 30 mg.kg⁻¹ every day) showed no significant differences. Body weight is expressed as weight evolution over the 21 day-treatment period. (B) The weight of heart, spleen, kidney, and liver was evaluated in mice daily injected with Triflorcas (30 mg.kg⁻¹) or vehicle for 21 days. No significant differences were observed. Values are expressed as means ± s.e.m. <i>P</i>>0.05. (C and D) Triflorcas treatment (i.p. 30 and 60 mg.kg⁻¹ every other day) reduced tumor volume (C) and weight (D) in nude mice injected sub-cutaneously with H1437 cells. Crizotinib (50 mg.kg⁻¹ daily) did not impair tumor growth. Values are reported as boxplots and expressed as means ± s.e.m. *<i>P</i><0.05; Student-<i>t</i> test.</p

Schematic representation of bioinformatic methodology applied to the NCI Anticancer Drug Screen outcomes.

<p>The scheme displays the bioinformatics strategy applied to compare the sensitivity profile of NCI cancer cells with changes in molecular signatures from NCI data resources. 1: Sensitiveness of NCI cell lines to Triflorcas was extracted by using the mean GI₅₀ into a numeric format and standard scores were visualized using green and red squares to indicate cells responding or not to Triflorcas, respectively. 2: NCI data resources applied to these studies (whole set of 87,545 measured targets) included: “Microarrays”, “all NCI dataset”, and “only Protein subset NCI” (see Material and Methods). The same color code was applied to indicate positive (green square) or negative (red square) changes of each molecular target analyzed.</p

Triflorcas alters the phosphorylation status of cell cycle-related proteins.

<p>(A) The phosphorylation status of several cell cycle proteins was analyzed by applying the phospho-array KPSS 10.1 (Kinexus Bioinformatics). GTL-16 cells were treated with vehicle (control) or Triflorcas (TFC; 3 µM) for 72 hours (left and right panels, respectively). Red circles highlight constituents of the Akt/mTOR/S6 pathway. Green and blue circles surround nucleophosmin/B23 and Rb phospho-epitopes, respectively. (B) The graph shows the ratio of phosphorylation levels of the indicated proteins in cells untreated versus those exposed to Triflorcas. (C) Phosphorylation levels of nucleophosmin/B23 at Ser₄ and Rb at S₇₈₀ were increased and decreased, respectively, in GTL-16 cells exposed to Triflorcas (TFC; 3 µM for 24 hours).</p

Triflorcas interferes with Met phosphorylation, its cellular localization, and activation of the PI3K/Akt pathway.

<p>(A) Met phosphorylation was analyzed by immuno-cytochemistry on H1437 cells untreated, treated with HGF, with Triflorcas (TFC; 10 µM for 24 hours) or with Triflorcas (10 µM for 24 hours) plus chlorpromazine (Chlor; 10 µg/ml for 2 hours) followed by HGF stimulation (20 ng/ml for 30 minutes). Triflorcas reduced the levels of Met phosphorylation induced by HGF. Note also that HGF-induced phospho-Met is localized at the plasma membrane of control cells, whereas it appears internalized in cells exposed to Triflorcas. Endocytosis inhibition with the chlorpromazine drug restored phospho-Met localization at the cellular membrane. Arrows indicate cluster of phosphorylated Met at the plasma membrane (40X magnification). (B) HGF-induced (20 ng/ml) phosphorylation levels of Met, Gab1, Akt, and p70^S6K were reduced in H1437 cells exposed to Triflorcas. In contrast, no changes were observed on ERKs phosphorylation levels. Similar expression levels of total Gab1, Akt, and p70^S6K were also found, indicating that Triflorcas interfered with their phosphorylation rather than with their expression levels. Western blot analyses were performed on total protein lysates. (C) Phosphorylation levels of Akt, p70^S6K, and S6 ribosomal protein, but not ERKs, were reduced in GTL-16 cells exposed to Triflorcas. Actin or Tubulin protein levels were used as loading controls in all experiments (lower panels in B and C). (D and E) Anchorage-independent growth of H1437 (D) and of GTL-16 (E) cells was impaired in the presence of Triflorcas (TFC), LY294002 (PI3K inhibitor), or A443654 (Akt inhibitor). Values are expressed as means ± s.e.m. **P<0.01; ***P<0.001; Student-<i>t</i> test.</p

Cytotoxic effects of Triflorcas on the NCI60 panel of cancer cell lines.

<p>The cytotoxic effects of Triflorcas (10, 100 nM, 1, 10, 100 µM) was determined by sulphorhodamineB assay after 48 hours of drug treatment. Results shown are representative of two independent experiments.</p