Plk1 inhibits the HIV-1 pseudovirus replication.

<p>(A) HCT116 cells were transfected with Plk1, Plk1 TD or Plk1 KD expression plasmids for 24 h and then infected with VSV-G pseudotyped pNL4.3-Luc virus for 24 h. The cell lysates were subjected to luciferase assay (left) and immunoblotting (right). “C” indicates that empty vector was used as the negative control. (B) 293T cells were transfected with Plk1-specific siRNA or control siRNA for 36 h. Then the cells were infected with VSV-G pseudotyped pNL4.3-Luc virus for 24 h. The cell lysates were harvested for luciferase assay (left) and immunoblotting (right). (C) HCT116 cells were infected with VSV-G pseudotyped pNL4.3-Luc virus for 24 h, then treated with BI2536, Nocodazole or DMSO for 16 h. The cell lysates were harvested for luciferase assay (left) and immunoblotting (right). The data are shown as the mean±SD from three independent experiments. Statistical significance was determined by Students t-test (*<i>p value</i> <0.05).</p

Identification of binding region of Plk1 and cyclin T1.

<p>(A) 293T cells were transfected with pCMV FLAG-Plk1or the indicated Plk1 deletion mutants, or empty vector as the control. The total cell extracts were immunoprecipitated with FLAG antibody and immunoblotted with Cdk9 and cyclin T1 antibody respectively. (B) GST pull-down assay. Equivalent amount of cell lysates from 293T cells were incubated with immobilized GST, GST-Plk1 polo-box domain (PBD) or GST-Plk1 PBD H538A/K540A, whose residues crucial for PBD phosphopeptide binding were mutated to alanine, followed by immunoblotting with Cdk9 and cyclin T1 antibody. (C) Structure of human cyclin T1 and cyclin T1 truncations. (D) GST-cyclin T1 and its truncations and His-Plk1 were purified and subjected to GST pull-down assay.</p

Plk1 interacts with P-TEFb independent of it kinase activity.

<p>(A) 293T cells were transfected with pCMV myc-Plk1 and pCMV FLAG-Cdk7 or FLAG-Cdk9, or empty vector as the control. The total cell extracts (TCE) were immunoprecipitated (IP) with myc antibody and immunoblotted (IB) with FLAG antibody. Asterisk indicates cross-reacting unrelated band. (B) 293T cells were transfected with empty vector or pCMV FLAG-Plk1. The total cell extracts were immunoprecipitated with FLAG antibody and immunoblotted with Cdk9 and cyclin T1 antibodies. The empty vector was used as the negative control. (C) HeLa cells were synchronized in M phase by Nocodazole treatment. The cell lysates were harvested and immunoprecipitated with normal mouse serum or Plk1 antibody, and then immunoblotted with Cdk9, cyclin T1 and Plk1 antibodies respectively. (D) GST pull-down assay. Purified His-Plk1 was incubated with immobilized GST, GST-cyclin T1 or GST-Cdk9 respectively. The bound protein was detected by immunoblotting with Plk1 antibody. <i>CBB</i>, Coomassie Brilliant Blue. (E) 293T cells were transfected with pCMV FLAG-Plk1, pCMV FLAG-Plk1 KD (K82R) or empty vector as the control. The cell lysates were immunoprecipitated with FLAG antibody and immunoblotted with Cdk9 and cyclin T1 antibodies.</p

Plk1 inhibites the kinase activity of the P-TEFb complex.

<p>(A) <i>In vitro</i> kinase assay. HCT116 cells transfected with pCMV FLAG-cyclin T1 were synchronized into different phases as described in Material and Method. The synchronization of the cells was detected by FACS. Cell lysates were immunoprecipated with FLAG antibody. Half of the immunoprecipates were subjected to immunoblotting with Cdk9 and FLAG antibody. The other half of the immunoprecipates were then incubated with GST-RNA Pol II CTD in the presence of [γ-³²P] ATP for the <i>in vitro</i> kinase assay. (B) FLAG-cyclin T1 over-expressed in 293T cells were immunoprecipated with FLAG antibody. The immunoprecipitated cyclin T1 complexes were preincubated with the lysates from HeLa cells either asynchronized or synchronized in M phase with cold ATP for the <i>in vitro</i> kinase assay, and washed with kinase buffer. Then the FLAG-cyclin T1 complexes were incubated with GST-RNA Pol II CTD and [γ-³²P] ATP for a second round of <i>in vitro</i> kinase assay. The expression level of Plk1, phosphorylation of histone H3 in HeLa cells and the immunoprecipitated FLAG-cyclin T1 after incubating with Hela cell extracts and washed were detected by immunoblotting with the indicated antibody. (C) 293T cells were transfected with pCMV FLAG-cyclin T1 or pCMV FLAG-Cdk9 respectively. The cell lysates were immunoprecipitated with FLAG antibody and the immunoprecipitated complexes were subjected to <i>in vitro</i> kinase assay in the presence or absence of His-Plk1 TD or His-Plk1 KD with GST- RNA Pol II CTD as the substrate. (D) pCMV FLAG-cyclin T1 transfected 293T cells were treated with or without BI2536(1µM) for 3.5 h before harvest. The FLAG-cyclin T1 complexes were immunoprecipated with FLAG antibody and subjected to <i>in vitro</i> kinase assay with GST-RNA Pol II CTD as the substrate. Equal amount of the immunoprecipated FLAG-cyclin T1 and Cdk9 was shown. (E) FLAG-tagged cyclin T1, cyclin T1 S564A, cyclin T1 S564D over-expressed in 293T cells were immunoprecipitated with FLAG antibody and incubated with GST- RNA Pol II CTD and [γ-³²P] ATP for the <i>in vitro</i> kinase assay. The immunoprecipated FLAG-cyclin T1 and Cdk9 were subjected to immunoblotting.</p

Plk1 represses P-TEFb-dependent transcription.

<p>(A) Schematic map of G5-83-HIV-Luc reporter. (B) 293T cells were co-transfected with Plk1, Plk1 TD or Plk1 KD expression plasmids and HIV-1 LTR luciferase reporter for 36 h. The cell lysates were harvested for luciferase assay. The expression level of Plk1 and its mutants was monitored by immunoblotting with FLAG antibody with β-actin as the internal control. (C) 293T cells were transfected with different doses of pCMV FLAG-Plk1 (150ng, 300ng and 600ng) and empty vector to keep an equal amount of co-tranfected DNA in each group, and with G5-83-HIV-luc luciferase reporter. After 36 h, the cell lysates were subjected to luciferase assay. The expression level of Plk1 was monitored by immunoblotting with FLAG antibody with β-actin as the internal control. (D) 293T cells were transfected with Plk1-specific siRNA or control siRNA followed by transfection with G5-83-HIV-luc luciferase reporter. The luciferase assay was performed 24 h after transfection. Immunoblotting was performed to detect the expression level of Plk1 and β-actin. (E) 293T cells were transfected with HIV-1 LTR luciferase reporter for 24 h and then treated with BI2536(100nM,500nM,1µM), Nocodazole or DMSO for 16hr before harvest. The cell lysates were subjected to luciferase assay and immunoblotting with Plk1 and β-actin. (F) NIH3T3 cells were transfected with human cyclin T1, cyclin T1 S564A, or cyclin T1 S564D expression plasmids with HIV-1 LTR reporter in the presence or absence of pCMV Tat for 24 h. The cell lysates were subjected to luciferase assay and immunoblotting with FLAG antibody and β-actin. The luciferase activity was normalized to the amount of luciferase DNA in transfected cells which was quantified by real-time PCR. The data are shown as the mean ± SD from three independent experiments. Statistical significance was determined by Students t-test (* <i>p value</i> < 0.05). “C” indicates that empty vector was used as the negative control.</p

Plk1 phosphorylates cyclin T1 at Ser564.

<p>(A) Purified GST or GST-cyclin T1 were incubated with His-Plk1 TD for the <i>in vitro</i> kinase assay. <i>CBB</i>, Coomassie Brilliant Blue. Asterisk indicates Plk1 autophosphorylation. (B) Identification of phosphorylation site(s) of cyclin T1 by HPLC-ESI/MS/MS spectrometry. Purified GST-cyclin T1 was incubated with His-Plk1 TD for the <i>in vitro</i> kinase assay in the presence of cold ATP and then subjected to mass spectrometry analysis. [y₉+HPO₃] indicates phosphorylation with an increasement of 80 mass unit. (C, D) Purified GST-cyclin T1 and GST-cyclin T1 S564A mutant (C) or GST-cyclin T1(531-630) and GST-cyclin T1(531-630) S564A (D) were incubated with His-Plk1 TD in the presence of [γ-³²P] ATP for the <i>in vitro</i> kinase assay. <i>CBB</i>, Coomassie Brilliant Blue staining. The phospho-signal was normalized to the total amount of GST-cyclin T1 or its mutants. (E) 293T cells were transfected with pCMV FLAG-cyclin T1 for 36 h and then treated with DMSO or BI2536 (1µM) for 3.5 h before harvest. Cell lysates were immunoprecipitated with FLAG antibody and followed by immunoblotting with anti-phospho-Serine and FLAG antibodies. The phospho-signal was normalized to the total amount of FLAG-cyclin T1. The quantification is represented as the mean ± SD from three independent experiments. Statistical significance was determined by Students t-test (<i>p value</i> < 0.05).</p

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