Co-immunoprecipitation and <i>in</i><i>vitro</i> phosphorylation of ESCRT proteins by human Plk1.

<p>(a) HEK293 cells were transfected with DNA for CHMP6-GFP, CHMP4B-YFP, CHMP3-myc, CHMP2A-GFP, or with Lipofectamine 2000 in the absence of transforming DNA. Lysates were prepared and anti-Plk1 antibody was added to each cell lysate. Complexes were dissociated from beads (<i>IP</i>: one-eighth was loaded). <i>Input</i> refers to the cell lysate that was interrogated: 10 µg was loaded. Experiments were repeated with qualitatively similar results. (b) MBP-His-ESCRT proteins were purified from <i>E. coli</i> and incubated at 30°C with recombinant Plk1 and [³²P-γ]-ATP for 0, 10 and 30 minutes. The reaction was stopped by the addition of Laemmli sample buffer. Samples were heated to 65°C, separated by SDS-PAGE and assayed by autoradiography. Shown is a typical experiment repeated three times using two different preparations of each recombinant protein and two different batches of recombinant Plk1. Coomassie Blue was used to confirm equal loading of recombinant ESCRT in each of the lanes. The major band at 65 kDa is BSA from the buffer used to resuspend Plk1. (c) MBP-His-Snf7p purified from <i>E. coli</i> and incubated at 30°C with or without recombinant Plk1 and [³²P-γ]-ATP for 30 minutes. Molecular weight markers are indicated.</p

ESCRT proteins and Plo1p, Ark1p and Clp1p are required for vacuolar sorting in fission yeast.

<p>(a) Defective vacuolar sorting is observed in fission yeast with individual chromosomal deletions of ESCRT genes. Wild-type and ESCRT-deleted fission yeast strains, transformed with Ub-GFP-<i>Sp</i>CPS, were cultured in liquid minimal medium at 25°C, stained with FM 4-64, and visualised using confocal microscopy. Mutants of <i>plo1</i> and <i>ark1</i> (b) and <i>clp1</i> (c) cause defective vacuolar sorting in fission yeast. Wild-type and fission yeast <i>plo1-ts35</i>, <i>ark1-T8</i>, <i>ark1-T11</i> and <i>clp1</i> strains, transformed with Ub-GFP-<i>Sp</i>CPS, were cultured in liquid minimal medium, stained with FM 4-64, and visualised using a confocal microscope. Strains were cultured at 25°C or 30°C. Scale bar, 10 µm. Experiments were performed three times with qualitatively similar results and data from a typical experiment are shown.</p

Genetic interactions between <i>plo1-ts35, ark1-T8</i> and <i>clp1Δ,</i> and ESCRT deletions in controlling vacuolar cell sorting in fission yeast.

<p>Double mutants of ESCRT deletions and (a) <i>plo1-ts35</i>, (b) <i>ark1-T8</i> or (c) <i>clp1Δ</i>, transformed with Ub-GFP-<i>Sp</i>CPS, were cultured in liquid minimal medium at 25°C, stained with FM 4-64, and visualised using confocal microscopy. Scale bar, 10 µm.</p

Physical interactions between ESCRT proteins and Plo1p as revealed by two-hybrid analysis.

<p>(a–e) Budding yeast strains containing <i>LEXA</i>–galactosidase transcriptional readout were transformed with the yeast two-hybrid bait (LexA DNA binding domain) and prey (GAL4 transcription activation domain) constructs indicated. Plo1p was fused to LexA and ESCRT proteins from each class were fused to GAL4. Mbx1p, a known Plo1p interacting protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111789#pone.0111789-Papadopoulou1" target="_blank">[17]</a>, was used a positive control. Interactions with mutants in Plo1p in the kinase domain (<i>K69R</i>) and polo boxes (<i>472–684</i> and <i>DHK625AAA</i>) were also performed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111789#pone.0111789-Reynolds1" target="_blank">[18]</a>. Transformed strains were grown for three days on selective medium with the X-gal overlay assay then performed. Experiments were performed three times with qualitatively similar results and illustrative examples of performed two-hybrid reactions are shown. (f) Summary of physical interactions between Plo1p and ESCRT proteins identified by yeast two-hybrid analysis. The table indicates the presence or absence of yeast showing a blue colour observed when each of the ESCRT proteins with various versions of Plo1p was assayed.</p

Physical interactions between ESCRT proteins and Ark1p as revealed by two-hybrid analysis.

<p>(a–e) Budding yeast strains containing <i>LEXA</i>–galactosidase transcriptional readout were transformed with the yeast two-hybrid bait (LexA DNA binding domain) and prey (GAL4 transcription activation domain) constructs indicated. Ark1p was fused to LexA and ESCRT proteins from each class were fused to GAL4. Mbx1p, a known Plo1p interacting protein,²⁸ was used a positive control for the assay. Transformed strains were grown for three days on selective medium with the X-gal overlay assay then performed. Experiments were performed three times with qualitatively similar results and illustrative examples of performed two-hybrid reactions are shown. (f) Summary of physical interactions between Ark1p and ESCRT proteins identified by yeast two-hybrid analysis. The table indicates the presence or absence of yeast showing a blue colour observed when each of the ESCRT proteins with various versions of Ark1p was assayed.</p

Synthetic septation phenotypes observed in double mutants in genes encoding ESCRT proteins and (a) <i>plo1-ts35</i>, (b) <i>ark1-T8</i> or (c) <i>clp1Δ</i>.

<p>Fission yeast double mutant strains were grown in complete liquid medium at 25°C to mid-exponential phase and harvested. Cells were stained with Calcofluor white and visualised using fluorescence microscopy. Both fluorescence and bright field images are shown. Scale bar, 10 µm. The frequency of phenotypes A–F (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111789#pone-0111789-g001" target="_blank">Figure 1a</a>) was quantitatively analysed in strains containing double mutants, in comparison to each parent. In each case 400 cells were counted in triplicate (*<i>p</i><0.05). Each of the ESCRT genes labels is accompanied by its respective ESCRT complex identification (<i>E-0</i>, <i>E-I</i>, <i>E-II</i> and <i>E-III</i>).</p

ESCRT proteins are required for septation in fission yeast.

<p>(a) Defective septation in fission yeast strains containing chromosomal deletions of ESCRT genes. Wild-type and strains containing individual chromosomal deletions of ESCRT genes were grown at 25°C in complete liquid medium to mid-exponential phase and harvested. Cells were stained with Calcofluor white and visualised using fluorescence microscopy. Both fluorescence and bright field images are shown. Panels A-F show representative cells illustrating observed septation phenotypes. Schematic diagrams above panels represent each phenotype: (A) a normal septum, (B) a misaligned septum, (C) a non-perpendicular septum, (D) multiple septa, (E) no septal formation, and (F) failed separation of daughter cells following septation. Scale bars, 10 µm. Data from a typical experiment repeated three times is shown. (b) Quantitative analysis of the frequency of septation phenotypes A–F in strains containing ESCRT chromosomal deletions, in comparison to wild-type. In each case, 400 cells were counted in triplicate. An asterisk (*) indicates a <i>p</i> value<0.05, indicating a significant difference to wild-type; n = 3. Each of the ESCRT gene labels is accompanied by its respective ESCRT complex identification (<i>E-0</i>, <i>E-I</i>, <i>E-II</i> and <i>E-III</i>).</p

Analysis of induction in cadmium chloride-treated cells transfected with TFBS-UR plasmids.

<p>HEK293 cells transfected with a plasmid pool, that included the plasmids listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s003" target="_blank">Table S2</a> and pRL-SV40 and were subsequently treated with cadmium. (A) Microarray-based detection of TF derived activation of UR expression. (B) qPCR-based detection of TF-derived activation of UR expression. Values are presented as log2 treatments of the fold induction of the TFBS-directed UR expression after treatment with the inducer of interest. The grey bar represents treatment-independent changes in the system. TFBS marked with * represent treatment-dependent effects on the TF library. Numerical data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s004" target="_blank">Table S3</a>. A statistical analysis of the qPCR assay data is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone-0050521-g003" target="_blank">Figure 3</a>.</p

Induction of the TF proteins of interest in HEK293 cells after treatment with forskolin, TPA and cadmium.

<p>Proteins extracted from treated and control cells were analyzed using Western blots and TF-specific antibodies. The levels of phosphorylated TFs and inactive TFs were analyzed for (A) CREB and ATF, (B) IκB, (C) c-jun and (D) SP1. Tubulin was used as a loading control. Quantification of the levels of protein on the Western blots showed a 1.6 and 1.3 fold increase in P-CREB and P-ATF after treatment with forskolin and a 1.5 and 1.6 fold increase in P-IκB, and P-c-jun after treatment with TPA. Treatment of HEK293 cells with cadmium chloride, dexamethasone, forskolin and TPA resulted in a 1.1, 1.1. 1.0 and 1.0 fold increase in the levels of SP1 protein. (E) Increased <i>hMTIIA</i> gene expression in HEK293 cells after treatment with cadmium. Expression of the cadmium-responsive <i>hMTIIa</i> gene was normalized to the expression of the chromosomal reference gene <i>B2M.</i> Abbreviations: -, carrier only control; C, cadmium; D, dexamethasone; F, forskolin; T, TPA.</p

Induction of selected TFBS-directed UR expression in HEK293 cells after treatment with cadmium, dexamethasone, TPA and forskolin.

<p>HEK293 cells transfected with a plasmid pool, that included the plasmids listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s003" target="_blank">Table S2</a> and pRL-SV40 and were subsequently treated with drugs of interest. (A) MRE-directed UR expression after treatment with cadmium. (B) GRE-directed UR expression after treatment with dexamethasone. (C) NF-κB-directed UR expression after treatment with TPA. (D) CREB-directed UR expression after treatment with forskolin. Values are presented as log2 treatments of the fold induction of the TFBS-directed UR expression after treatment with the inducer of interest. The error bars are calculated as 1 standard error of the mean each way.</p