MCC cell lines exhibit variable ECAR and sensitivity to MCT1 inhibition.

<p><b>A)</b> ECAR (mpH/min) of MKL-1, MKL-2 and WaGa lines (minutes). Cells were treated with oligomycin (1 μM) at the indicated time point (arrow). <b>B-D)</b> XTT proliferation assay of MKL-1, MKL-2 and WaGa cells treated with either DMSO, CHC (5 mM), SR13800 (100 nM), or SR13801 (100 nM) (days).</p

Temporal transcriptome of IMR90 fibroblasts inducibly expressing MCPyV ST.

<p><b>A)</b> IMR90 fibroblasts containing dox-inducible MCPyV ST or GFP vectors were treated with dox and harvested every 8 hours for RNA extraction. Each time point represents three biological replicas. <b>B)</b> Mean ST transcript levels and <b>C)</b> immunoblotting for ST, GFP and vinculin from cells collected every 8 hours for 96 hours following dox treatment. <b>D)</b> Hierarchical clustering and fold change between MCPyV ST and GFP following dox induction for 96 hours. Each bar represents an average of three experiments for each time point. The enrichment of “Cancer Hallmark” gene sets are represented relative to the ST-differentially expressed clusters, including epithelial to mesenchymal transition (EMT), tumor necrosis factor-α (TNFA signaling via NF-κB), hypoxia, mTORC1, oxidative phosphorylation, glycolysis, MYC, and several cell cycle clusters including E2F targets, G2M checkpoint and mitotic spindle. The color bar indicates statistical significance, yellow p < 0.05 and gray p > 0.05.</p

MCPyV ST increases aerobic glycolysis and MCT1 sensitivity.

<p><b>A)</b> Media glucose (Glc) and lactate (Lac) levels (mM) from cultures of IMR90 cells expressing ST or GFP were measured at the indicated day following dox addition. **P < 0.005 calculated using unpaired student’s T test between the marked GFP and ST points. <b>B)</b> ECAR (mpH/min) of IMR90 cells inducibly expressing ST with and without dox addition for 48 hours. ***P < 0.0005 calculated using unpaired student’s T test. <b>C)</b> ECAR of IMR90 cells expressing ST or GFP with CHC (5 mM) or DMSO (minutes) following 48 hours of dox treatment. Cells were treated with oligomycin (1 μM) at the indicated time point. ***P < 0.0005 calculated using unpaired student’s T test between GFP-DMSO and ST-DMSO samples. <b>D)</b> OCR (pmoles/min) of cells (minutes) as in C. <b>E)</b> Growth of IMR90 cells expressing ST or GFP treated with dox and CHC or DMSO was assessed by crystal violet every day for 5 days. ***P < 0.0005 calculated using unpaired, two-tailed student’s T test between ST-DMSO and ST-CHC treatments. Key same as in C.</p

MYC isoforms differentially regulate glycolysis gene expression and ECAR of MCC cells.

<p><b>A)</b> MKL-1 and WaGa cells containing inducible vectors for MYC, MYCN or MYCL were treated with (+) or without (-) dox for 72 hours and lysates were immunoblotted with the indicated antibodies. <b>B)</b> ECAR (mpH/min) of MKL-1 cells inducibly expressing GFP, MYC, MYCN or MYCL after 72 hours of dox addition (minutes). Cells were treated with oligomycin (1 μM) at the indicated time point. *P < 0.05 calculated using unpaired student’s T test between MYC and MYCL samples.</p

MCPyV-transformed cells exhibit elevated ECAR and sensitivity to MCT1 inhibitors.

<p><b>A)</b> Anchorage-independent growth of IMR90 PH, PHL, PHE and PHEL cells. ****P < 0.0001 calculated using ordinary one-way ANOVA with multiple comparisons. <b>B)</b> Basal ECAR (mpH/min) measurement of p53DD, PH, PHE and PHE + MYCL (PHEL) cells. **P < 0.005 and ****P < 0.0001 calculated using ordinary one-way ANOVA with multiple comparisons. <b>C)</b> Proliferation of PHEL cells treated with DMSO, CHC (5 mM), SR13800 (100 nM), or SR13801 (100 nM) was assessed by crystal violet staining. *P < 0.05 calculated using student’s T test between DMSO-SR13800 and DMSO-SR13801 samples. <b>D)</b> Anchorage-independent growth of IMR90 PHE and PHEL cells treated with DMSO, CHC, SR13800 (SR800) or SR13801 (SR801). ****P < 0.0001 calculated using ordinary one-way ANOVA with multiple comparisons.</p

Generation of tagged cyclin E1 knock-In mice and analyses of cyclin E1-containing protein complexes.

<p>(A and B) Targeting strategy to knock-in Flag and HA tags into <i>the cyclin E1</i> locus to generate N-terminally tagged <i>cyclin E1</i>^<i>Ntag</i> (A) and C-terminally tagged <i>cyclin E1</i>^<i>Ctag</i> alleles (B). The exons are shown as green boxes, Flag tag as a blue box, and HA tag as a red box. Start and stop codons are marked with orange and yellow arrowheads, respectively. The hygromycin resistance cassette (Hyg) with flanking loxP sequences (filled arrows) is also indicated. Restriction enzyme recognition sites: E, EcoRI; A, AflII; Sc, ScaI; N, NotI; X, XhoI; K, KpnI; S, SpeI; P, PmeI; Hp, HpaI. Note that panel (A) was shown in ref [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (C) Western blot analysis of wild-type control (Ctrl), heterozygous cyclin <i>E1</i>^{<i>+/Ntag</i>}, <i>cyclin E1</i>^{<i>+/Ctag</i>}, and <i>cyclin E1</i>^{<i>Ntag/Ctag</i>} embryonic stem cells probed with anti-cyclin E1 and -HA antibodies. Actin served as a loading control. Forth panel: cyclin E1 was immunoprecipitated with anti-Flag antibody and the immunoblots were probed with anti-Cdk2 antibody. Fifth panel: anti-Flag immunoprecipitates were used for <i>in vitro</i> kinase reactions using histone H1 as a substrate. (D) Same analyses as in (C) using spleens of homozygous knock-in mice. Lanes 1–2 in panels (C and D) were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (E) Cyclin E levels detected by western blotting in the indicated organs of 1-month-old mice and in embryonic brain (day E14.5). Actin served as a loading control. The last two lanes (Brain) were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (F) Quantification of cyclin E levels in different organs, normalized against actin (from E). (G) Protein lysates from brains and testes of adult tagged cyclin E1 knock-in mice were separated by gel-filtration chromatography. Fractions containing protein complexes of the indicated molecular weights were analyzed by western blotting for cyclin E using an anti-HA antibody. (H) Cyclin E1-associated proteins were purified from the indicated organs of tagged cyclin E1 knock-in (KI) mice, or from control mice (Ctrl, ‘mock’ purifications) by sequential immunoaffinity purifications with anti-Flag and -HA antibodies, and 10% of the final eluate was resolved on PAGE gels and silver-stained. Arrows indicate bands corresponding to cyclin E1. Panels representing embryonic and adult brains were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>].</p

Interaction of cyclin E1 with the DREAM complex.

<p>(A) A diagram showing in which organ a particular component of the DREAM complex was identified as a cyclin E1-interacting protein by our mass spectrometric analyses. The number of organs in which a given protein was found to associate with cyclin E1 is depicted by the color. (B) Association of cyclin E1 with components of the DREAM complex in the spleens of tagged knock-in mice (KI) was verified by immunoprecipitating (IP) cyclin E1 with anti-Flag antibody followed by immunoblotting with the indicated antibodies. (C) IP followed by re-IP-immunoblotting to demonstrate that cyclin E1, Cdk2 and the DREAM complex components are present within the same multi-protein complex. Cyclin E1 was immunoprecipitated from spleens of KI mice using anti-Flag antibody, protein complexes were eluted with Flag peptides, re-immunoprecipitated with IgG (control) or with anti-p130 antibody, and then immunoblotted with the indicated antibodies. (D) T98G cells were serum starved for 72 hrs (0% FBS). Subsequently, cells were stimulated to re-enter the cell cycle by addition of 20% FBS supplemented with either 0.2% DMSO (control, left two panels) or 20 μM CVT-313 (right two panels), and harvested at the indicated time-points. Cell extracts (whole) as well as anti-Lin37 immunoprecipitates were resolved on 4–15% gradient SDS-PAGE gels and probed with indicated antibodies. Gapdh serves as a loading control. (E) Cyclin E1-Cdk2 kinase can phosphorylate purified recombinant Lin proteins <i>in</i> vitro. Lin9, Lin52 and Lin54 were expressed as GST-fusion proteins in <i>E</i>. <i>Coli</i>, purified and subjected to <i>in vitro</i> kinase reactions with the recombinant cyclin E1-Cdk2 in the presence of [γ³²P]ATP. Recombinant histone H1 was used as a positive control and GST as a negative control. (F) A diagram illustrating amino acid residues in human Lin proteins that were phosphorylated by cyclin E-Cdk2. (G) Wild-type Cdk2 (K2WT) or analog-sensitive Cdk2 (K2AS) were transfected into 293T cells together with cyclin E1 and Flag-tagged Lin37 or vector control. After supplementing cells with 6-Fu-ATPγS, labeling of Lin37 was evaluated by immunoprecipitating Lin37 with an anti-Flag antibody followed by immunoblotting with anti-thiophosphate ester antibody. A blue arrowhead indicates 6-Fu-ATPγS-labeled Lin37.</p

Identification of Mybl1 and Dmrtc2 as cyclin E-Cdk2 phosphorylation substrates in the testes.

<p>(A) A diagram illustrating cyclin E1-interactome in testes, consisting of highest-confidence ‘core’ interactors (green nodes), and lower-confidence Category 3 interactors (blue nodes) that were included to the interactome based on their reported interaction with core interactors in the STRING database (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Solid lines depict STRING-verified interactions. Dashed lines depict an interaction derived from our mass spectrometry analyses between cyclin E1 and a protein that has no known interactions with other ‘core’ interactors. Red arrows indicate proteins that were previously implicated to play important roles in spermatogenesis. (B) Interaction between endogenous Cdk2/cyclin E and Mybl1 and Dmrtc2 in mouse testes, detected by IP–western blotting. Cdk2 or Mybl1 were immunoprecipitated from lysates of testes, and immunoblots were probed with the indicated antibodies. (C) N-terminal fragment (aa 1–201) of Dmrtc2, as well as full length, N-terminal (aa 1–376), and C-terminal (aa 376–752) fragments of Mybl1 were expressed as GST-fusion proteins in <i>E</i>. <i>Coli</i>, purified and subjected to <i>in vitro</i> kinase reactions with the recombinant cyclin E1-Cdk2 in the presence of [γ ³²P]ATP. Recombinant histone H1 was used as a positive control and GST as a negative control. Red arrowheads point to phosphorylated GST-fusion proteins, orange arrowheads indicate phosphorylated truncated proteins, and blue arrow indicates auto-phosphorylated recombinant cyclin E1-Cdk2. (D) Wild-type Cdk2 (K2WT) or analog-sensitive Cdk2 (K2AS) were transfected into 293T cells together with cyclin E1 and Flag-tagged substrates (Mybl1 or Dmrtc2). After supplementing cells with 6-Fu-ATPγS, labeling of substrates was evaluated by immunoprecipitating Mybl1 or Dmrtc2 with anti-Flag antibody followed by immunoblotting with an anti-thiophosphate ester antibody. Blue arrowheads indicate ATPγS-labeled Mybl1 and Dmrtc2. The experiment was performed on the same gel as the one shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.g005" target="_blank">Fig 5G</a>, hence the vector control (Vector) is identical. (E) A diagram illustrating amino acid residues in Mybl1 and Dmrtc2, which were phosphorylated by cyclin E-Cdk2.</p

Quantitative proteomic (iTRAQ) analysis of cyclin E1-interacting proteins in mouse organs in the absence of Cdk2.

<p>(A) Relative abundance of cyclin E1-associated Cdk1, Cdk2, Cdk4, Cdk5 and p107 in the spleens of <i>Cdk2</i>^<i>-/-</i><i>/cyclin E1</i>^{<i>Ntag/Ntag</i>} mice, as compared to <i>Cdk2</i>^<i>+/+</i><i>/cyclin E1</i>^{<i>Ntag/Ntag</i>} animals, was determined by iTRAQ labeling and LC-MS. (B) The amount of cyclin E1-associated Cdk1, Cdk2, Cdk4, Cdk5 and p107 in the spleens of wild-type (Ctrl), <i>Cdk2</i>^<i>+/+</i><i>/cyclin E1</i>^{<i>Ntag/Ntag</i>} (KI), and <i>Cdk2</i>^<i>-/-</i><i>/cyclin E1</i>^{<i>Ntag/Ntag</i>} (Cdk2^<i>-/-</i>) mice was gauged by immunoprecipitation with an anti-Flag antibody and immunoblotting with the indicated antibodies. Abundance of each protein in total lysates (whole) is also shown. (C) Spleens from wild-type mice were incubated with 20 μM CVT-313 (+) or with vehicle only (-). Association of cyclin E1 with Cdk2, Cdk1, Cdk4 and Cdk5 was assessed by IP–western blotting. Whole, whole cell lysate from vehicle only-treated mice. Lower panel: To ensure that CVT-313 treatment inhibited Cdk2 kinase activity, Cdk2 was immunoprecipitated from lysates and used for <i>in vitro</i> kinase reactions with histone H1 as a substrate. Note that CVT-313 treatment strongly decreased Cdk2 kinase activity.</p

Cyclin E1-interactomes.

<p>(A) Diagrams depicting cyclin E1-interacting proteins in the indicated mouse organs. Cyclin E1 is shown as a red node. Green nodes denote highest-confidence ‘core’ interactors (Category 1, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Yellow and blue nodes represent, respectively, lower confidence Categories 2 and 3 interactors that were included to the interactome based on their reported interaction with core interactors in the STRING database (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Solid lines depict STRING-verified interactions. Dashed lines depict an interaction derived from our mass spectrometry analyses between cyclin E1 and a protein that has no known interactions with other core interactors. (B) A combined diagram depicting cyclin E1-interacting proteins from all five organs analyzed. Cyclin E1 is shown as a red node. Green nodes denote highest-confidence core (Category 1) interactors. Yellow and blue nodes denote, respectively, Categories 2 and 3 interactors, which were included into the interactome based on their ability to interact with core interactors as revealed by STRING (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Solid blue lines depict STRING-verified interactions between pairs of proteins that were identified by us as cyclin E1-interacting proteins within the same organ. Gray dotted lines depict STRING-verified interactions between pairs of proteins identified as cyclin E1-interators in different organs. Blue dashed lines depict interactions detected in our mass spectrometry analyses between cyclin E1 and a protein that has no known interactions with other core proteins within the same organ interactome.</p