Additional file 2: of ELF5 isoform expression is tissue-specific and significantly altered in cancer

Excel spreadsheet with details of all qPCR assays (Roche UPL assays worksheet 1 and TaqMan assays worksheet 2). (XLSX 25 kb

Anchorage independent cell growth of ChoKα1- and β1-overexpressing cells.

<p><b>A)</b> and <b>B)</b><i>In vitro</i> ChoK activity of cell-free extracts from transfected cells at the moment of plating, determined as conversion of ¹⁴C-labeled choline to PCho. <b>C)</b> Photographs of a representative experiment of the soft agar assay. A total of 10⁵ cells were plated per 60-mm dish, and the number of colonies quantified after 5-8 weeks of incubation. <b>D)</b> and <b>E)</b> Computer based automatic quantification of the number of colonies, mean values±SEM is represented. The assay was performed 3 independent times with triplicate samples obtaining similar results. Statistical significance (p≤0.05) is marked by an asterisk.</p

Differential activation of choline kinase α1 and β1 isoforms by Ras and Rho GTPases.

<p>Choline kinase isoforms were expressed alone or in combination with the indicated Ras and Rho GTPases and the <i>in vitro</i> ChoK activity determined. <b>A)</b> Analysis of ectopic expression by Western Blot in HEK293T transfected cell extracts of ChoKα1 (52 KDa), ChoKβ1(45 KDa), RhoA-QL(22 KDa), Cdc42-QL(25 KDa) and H-rasV12 (23 KDa). Empty vectors were used as controls for the endogenous levels, and GAPDH as loading control. <b>B)</b> and <b>C)</b><i>In vitro</i> choline kinase activity of ChoKα1 or ChoKβ1 in the presence of enhanced expression of constitutive active forms of RhoA, Cdc42 or H-Ras. <b>D)</b> and <b>E)</b><i>In vitro</i> ethanolamine kinase activity of ChoKα or ChoKβ in the presence of each indicated constitutive active form of GTPase. The results are represented as fold induction of conversion to the corresponding phosphorylated metabolite determined as total cpm/µg of whole cellular extract, and normalized to the empty vector transfected cells as control. Data shown represent the mean values±SEM of 3 independent experiments, each one performed with duplicate samples. Statistical significance (p≤0.05) is marked by an asterisk comparing to the activity achieved when ChoKα1 or ChoKβ1, where appropriate, are transfected alone.</p

Overexpression of ChoKβ1 is not sufficient to induce tumor growth in athymic nude mice.

<p>Xenografts were established by s.c. injection of transfected HEK293T or MDCK cells in athymic nu/nu nude mice. <b>A)</b> and <b>B)</b> Western Blot analysis of ectopic expression of choline kinase isoforms in transfected HEK293T or MDCK cells, respectively, before mice inoculation. <b>C)</b> and <b>D)</b> Analysis of choline kinase activity in ChoKα1 or β1 transfected HEK293T or MDCK cells-free extracts before mice inoculation. <b>E)</b> and <b>F)</b> Volume of tumors generated by subcutaneous injection of 10⁶ transfected cells. Tumoral volume was calculated according to the formula: <i>Vol = [D * d²]/2</i>, where D and d are major and minor tumor diameters respectively. The data from HEK293T represents mean values±SEM from two independent experiments (n₁ = 12; n₂ = 16), the MDCK experiment correspond to an equivalent experiment with n = 12.</p

Comparative expression of ChoKα1 and ChoKβ1 mRNA in a panel of cancer cell lines.

<p>Q-PCR was performed to determine the level of expression of mRNA in non-tumorogenic mammary cell lines (HMEC, MCF10A), breast cancer cell lines (MDA-MB435, MDA-MB468, T47D, MCF7), non-tumorogenic lung cells (BEC) and lung cancer cell lines (H510, H82). The data were normalized with the endogenous 18S ribosomal RNA. For the comparison between tumoral and non-tumoral cell lines, the 2^−ΔΔCt method was applied and log₁₀ RQ is represented. Note that the data are referred to the Human Mammary Epithelial Cells (HMEC) mRNA levels in breast cell lines and no significant difference in the level of both ChoK isoforms mRNA was found with the normal MCF10A cell line. The reference for lung cancer cells was the primary Bronchial Epithelial Cells (BEC).</p

Michaelis constant (Km) of ChoKα and β isoforms for choline and ethanolamine.

1<p>Data are represented in milliMolar.</p>2<p>Referenced to the lowest Km.</p><p>Km of the different isoforms of ChoK for each substrate is indicated in each case. The results were obtained from four independent experiments using the logarithmic Michaelis-Menten formula as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007819#s4" target="_blank">Material and Methods</a>.</p

Characterization of choline and ethanolamine kinase activity of ChoKα1 and Chokβ1 in HEK293T cells.

<p>HEK293T cells were transfected with eukaryotic expression vectors of human ChoKα1 and ChoKβ1 gene. pCDNA3b empty vector was used as control. <b>A)</b> Overexpression of ChoKα1 and ChoKβ1 in HEK293T cells detected by Western Blot. GAPDH detection was used as control of expression level. <b>B, C)</b> In vitro ChoK (B) and EtnK (C) activity of choline kinase α1 and β1 isoforms in cell-free extracts of HEK293T transfected cells. Percentage of conversion of ¹⁴C-choline or ¹⁴C-ethanolamine to the phosphorylated product is represented. The experiment was performed in duplicate samples, repeated 4 times, and mean±SEM values from all experiments estimated.</p

Additional file 8: Figure S3. of MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib

showing that MCL-1 antagonism by BIMs2A slows tumor growth in mice bearing MDA-MB-468-2A xenografts but not MDA-MB-231-2A xenografts. (A–F) Line graphs depicting the tumor growth curves of MDA-MB-468-2A xenografts (A, B) and MDA-MB-231-2A xenografts (C, D) from mice fed with DOX or control food. Linear regression of these curves shown in B and D respectively. A comparison of the growth rate of tumors in mice bearing MDA-MB-468-2A (black) and MDA-MB-231-2A (red) xenografts fed with control food (E, F). (JPG 1348 kb

Additional file 5: Figure S5. of MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib

showing that MCL-1 antagonism resulted in changes in proteins involved in SRC family kinase signaling and phosphorylation at serine3 of Cofilin. (A) Normalized z-ratio (a measure of statistical significance) of phosphorylated and total proteins (as indicated) in MDA-MB-468-2A cells at 24 hours after treatment with DOX compared with control cells. (B) Western blots of serine 3 phosphorylated Cofilin, total Cofilin and Actin in xenografts of MDA-MB-468-2A and MDA-MB-231-2A fed DOX food as indicated. (C) Bar graphs depicting the ratio of serine 3 phosphorylated Cofilin to total Cofilin from (B). Bars indicate statistically significant groups, Mann–Whitney p value. (D) Immunofluorescence of Cofilin and p-Cofilin MDA-MB-231-2A cells grown on fibronectin 24 hours after DOX or vehicle treatment. (E) Proximity ligation assays using antibodies to MCL-1 and Cofilin (green), Phalloidin (red) and Dapi (blue) in MDA-MB-231-2A cells. (JPG 1419 kb

Additional file 6: Figure S6. of MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib

showing that MCL-1 antagonism and Dasatinib treatment induced apoptosis in MDA-MB-468-2A cells but not MDA-MB-231-2A cells when grown in 2D monolayer cultures. Bar graphs depicting the average fraction of apoptotic cells (total Annexin V-positive by flow cytometry) as indicated after 24 hours after treatment with vehicle, DOX, 5 μM A1210477 and 5 μM UMI-77 alone and in combination with 1 μM dasatinib after 24 hours. All graphs and western blots are the average of three independent experiments. Bars indicate statistically significant groups, p value paired t tests. (JPG 1029 kb