Journal: PLoS ONE

Article Title: P-TEFb Kinase Activity Is Essential for Global Transcription, Resumption of Meiosis and Embryonic Genome Activation in Pig

doi: 10.1371/journal.pone.0152254

Figure Lengend Snippet: A) CDK9, Cyclin T1 and Cyclin T2 are present and located predominantly in the nuclei of pig GV oocytes. CDK9 and Cyclin T1 also show speckles in GV oocytes. Rabbit polyclonal antibodies were used for immunostaining. B) CDK9 and Cyclin T1 speckle-like structures are colocalized with Pol II CTD Ser2p in GV of NSN and pNSN oocytes. In SN oocytes, CDK9 tends to accumulate at the periphery of the NLB and also dissociate from Pol II CTD Ser2p speckles. C) CDK9 also colocalizes with the splicing speckle marker SC-35 protein (red) in GV of pNSN oocytes. In transcriptionally inactive SN oocytes, SC-35 is dispersed throughout the nucleoplasm but CDK9 aggregates around the NLB. SC-35 is immunostained with a mouse monoclonal antibody. DNA is counterstained with DAPI.

Article Snippet: CDK9 Inhibitor II/CAN508 (sc-203326) was purchased from Santa Cruz and dissolved in DMSO to form 5 mM stock solution.

Techniques: Immunostaining, Marker

Journal: PLoS ONE

Article Title: P-TEFb Kinase Activity Is Essential for Global Transcription, Resumption of Meiosis and Embryonic Genome Activation in Pig

doi: 10.1371/journal.pone.0152254

Figure Lengend Snippet: A) CDK9 is colocalized with the DFC component, fibrillarin, only at the periphery of the NLB in NSN oocytes. In pNSN oocytes, nucleolar structure is changed and fibrillarin and other nucleolar components move to the periphery of the NLB. In this stage, CDK9 shows partial colocalization with fibrillarin (inset). In SN oocytes, fibrillarin is mostly gone, but CDK9 remains bound to the nucleolar structure. At the periphery of the NLB, CDK9 shows more colocalization with the FC component, UBF, in pNSN (inset). B) The same pNSN oocytes as in A was analyzed further. Eight optical sections, Z-stacks with 0.5 μm, were taken by confocal microscope confirming the colocalization of CDK9 and fibrillarin at the periphery of NLB. C) X and Y axes of a single focal plane of the same image as B focusing on an area corresponding to the NLB periphery, indicating the spatially colocalization of CDK9 and fibrillarin in that area. D) Cyclin T1 also shows colocalization with nucleolar factor, UBF, in pNSN and pSN oocytes. DNA counterstained with DAPI. E) COCs were collected separately from small or large follicles and then were denuded and subjected to Western blotting. The same anti-CDK9 antibody as used for immunostaining was used for Western blot. The bands correspond to two CDK9 isoforms, 42 kD and 55 kD. Fifty oocytes were collected for each group.

Article Snippet: CDK9 Inhibitor II/CAN508 (sc-203326) was purchased from Santa Cruz and dissolved in DMSO to form 5 mM stock solution.

Techniques: Microscopy, Western Blot, Immunostaining

Journal: PLoS ONE

Article Title: P-TEFb Kinase Activity Is Essential for Global Transcription, Resumption of Meiosis and Embryonic Genome Activation in Pig

doi: 10.1371/journal.pone.0152254

Figure Lengend Snippet: A) COCs were collected from different size of follicles and immediately cultured in a complete maturation medium in presence of 5 mM FU for 1 hour. Then, COCs were denuded and oocytes were fixed and subjected to immunostaining. In NSN oocytes in which CDK9 is distributed throughout the GV, transcriptional activity is at the highest level. In SN oocytes, CDK9 accumulates around the NLB and nascent RNAs become undetectable. B) COCs were subjected or not to α-amanitin, actinomycin D (ActD), or Flavopiridol for 1 hour. Treatment with α-amanitin started 1 hour ahead of the other compounds. At the same time, nascent transcripts were labeled with FU. Then, COCs were denuded and oocytes were fixed and subjected to immunostaining. In α-amanitin-treated oocytes, nucleoplasmic transcription decreased dramatically, but a rim of transcripts remained around the nucleolus. In oocytes treated with ActD, no transcriptional activity was detected. In Flavopiridol-treated oocytes, similarly, almost all the RNA transcription was abolished. Similar result was obtained when oocytes treated with CDK9 Inhibitor II (CAN508).

Article Snippet: CDK9 Inhibitor II/CAN508 (sc-203326) was purchased from Santa Cruz and dissolved in DMSO to form 5 mM stock solution.

Techniques: Cell Culture, Immunostaining, Activity Assay, Labeling

Journal: PLoS ONE

Article Title: P-TEFb Kinase Activity Is Essential for Global Transcription, Resumption of Meiosis and Embryonic Genome Activation in Pig

doi: 10.1371/journal.pone.0152254

Figure Lengend Snippet: COCs were obtained from different size of antral follicles and immediately were treated or not with Flavopiridol or CAN508 for 1 hour in complete maturation medium. Then, they were denuded, fixed and subjected to RNA-FISH. Two fluorophore-conjugated DNA oligonucleotides were designed to probe for two distant regions of pre-rRNAs. One Cy3-conjugated probe recognized the 5’ external transcribed spacer (5’ETS, red) and the other, FAM-conjugated probe recognized the 28S rRNA (green) at the 3’ end of the pre-rRNA. In control group, the level and localization of 5’ETS and 28S in oocytes with different chromatin configuration was analyzed. While in NSN oocytes, signals from both probes were robust and centralized inside the NLB, in SN oocytes no signals were detected from either probe. Inhibition of CDK9 by Flavopiridol or by CAN508 essentially abolished the signals from both probes in the majority of transcriptionally active oocytes.

Article Snippet: CDK9 Inhibitor II/CAN508 (sc-203326) was purchased from Santa Cruz and dissolved in DMSO to form 5 mM stock solution.

Techniques: Control, Inhibition

Journal: PLoS ONE

Article Title: P-TEFb Kinase Activity Is Essential for Global Transcription, Resumption of Meiosis and Embryonic Genome Activation in Pig

doi: 10.1371/journal.pone.0152254

Figure Lengend Snippet: A) COCs were obtained from small and medium size antral follicles and matured in absence or presence of increasing concentration of Flavopiridol or CAN508. Then COCs were denuded and fixed and subjected to immunostaining against nuclear lamin A/C. DNA counterstained with DAPI. Oocytes with two distinct metaphase chromosomes and polar body were considered as MII. Oocytes with only one metaphase chromosome and no polar body were considered as MI. Oocytes with intact germinal vesicle and positive lamin A/C staining were considered as GV. The figure shows that inhibition of CDK9 strictly inhibits GVBD in pig oocytes. B) COCs were obtained from large antral follicles and treated or not with CDK inhibitors, Flavopiridol, Dinaciclib and JNJ-7706621 for 6 hours in complete maturation medium. Then, COCs were denuded and subjected to immunostaining. MPF/CDK1 kinase activity was measured using a monoclonal antibody recognizing the proteins that share two peptide motives LTPLK and FTPLQ. CDK1 phosphorylates the Thr residue of these motives and promotes GVBD. MPM-2 (green) recognizes these motives only when they are phosphorylated. Although treatment with Flavopiridol blocked GVBD, but the level of MPM-2 signal did not changed significantly compared with untreated oocytes. On the other hand, both Dinaciclib and JNJ-7706621 (JNJ) decline the level of CDK1 kinase activity. C) CDK1 activity is inhibited by phosphorylation of Thr14/Tyr15 residues. Dephosphorylation of these residues activates CDK1 and promotes GVBD. In Flavopiridol-treated oocytes, the level of pCDK1 did not change compared with the control group, but Dinaciclib, and less potently JNJ, elevated the level of inhibitory phosphorylation of CDK1. In all groups, the level of pCDK1 was normalized with pan CDK1. D) COCs were obtained from small or medium size antral follicles. Inhibition of CDK9 by Flavopiridol or Dinaciclib inhibited transcriptional activity of GV oocytes, but inhibition of CDK1 by JNJ did not changed the level of nascent RNAs compared with untreated oocytes. These experiments totally showed that GVBD block by Flavopiridol was due to inhibition of transcription but not inhibition of CDK1 activity.

Article Snippet: CDK9 Inhibitor II/CAN508 (sc-203326) was purchased from Santa Cruz and dissolved in DMSO to form 5 mM stock solution.

Techniques: Concentration Assay, Immunostaining, Staining, Inhibition, Activity Assay, Residue, Phospho-proteomics, De-Phosphorylation Assay, Control, Blocking Assay

Journal: PLoS ONE

Article Title: P-TEFb Kinase Activity Is Essential for Global Transcription, Resumption of Meiosis and Embryonic Genome Activation in Pig

doi: 10.1371/journal.pone.0152254

Figure Lengend Snippet: Confocal immunofluorescence analysis of CDK9 in whole-mount IVF and parthenogenetic embryos. A) CDK9 nuclear localization in IVF embryos. Pol II (red) was used as the nuclear marker. No intense signal at the periphery of the nucleolar precursor bodies was observed for CDK9. B) CDK9 nuclear localization in parthenogenetic embryos. Nucleolin (red) was used as the nucleolar marker. In 1-cell embryos, CDK9 was evenly distributed throughout pronuclei. At the 2-cell stage, CDK9 showed some localization at the periphery of NPBs in some cases. In late 4-cell embryos, CDK9 exhibited more profound nucleolar association and colocalized with nucleolin. As nucleolar precursors transformed to matured nucleoli in morula and blastocysts, CDK9 localized within the nucleoli with and surrounded by nucleolin. DNA was counterstained with DAPI. Enlarged views of the small insets are shown in the right panels. C) Embryos were produced via either IVF or parthenogenesis and cultured absence or presence of Flavopiridol (100nM) or CAN508 (10 μM) for 7 days. Then, the embryos were counterstained with DAPI and were analyzed under a fluorescent microscope. Embryos with zero nuclei were excluded. The first cleavage rate was not significantly changed in treated embryos. However, the majority of treated embryos tended to remain at 4-cell stage. Also, progress to next cell cycles was very poor in treated embryos compared with their control groups. No blastocyst was observed in groups treated with either CDK9 inhibitor.

Article Snippet: CDK9 Inhibitor II/CAN508 (sc-203326) was purchased from Santa Cruz and dissolved in DMSO to form 5 mM stock solution.

Techniques: Immunofluorescence, Marker, Transformation Assay, Produced, Cell Culture, Microscopy, Control

Journal: PLoS ONE

Article Title: P-TEFb Kinase Activity Is Essential for Global Transcription, Resumption of Meiosis and Embryonic Genome Activation in Pig

doi: 10.1371/journal.pone.0152254

Figure Lengend Snippet: A) The susceptibility of FU labeling was determined in preimplantation embryos. Embryos were cultured in PZM-3 medium. For nascent RNA labeling, embryos were transferred to the same medium plus 5mM FU and cultured for additional 1 hour. Then embryos were fixed and subjected to immunostaining. While 2-cell embryos showed very faint, if any, nascent RNA level, 4-cell stage embryos and blastocysts exhibited strong nuclear signals from FU incorporation into nascent RNAs. B) IVF embryos were cultured in PZM-3 medium in presence or absence of 100 nM Flavopiridol. One hour before fixation, embryos were transferred to the same media supplemented with FU. CDK9 inhibition dramatically decreased the level of nascent RNAs in late 4-cell stage embryos. The level of nuclear CDK9 also decreased slightly in these embryos. C) Treatment of late 4-cell embryos for 1 hour with Flavopiridol abolished the pre-rRNA transcription. Although the signal corresponding to both 5’ETS and 28S rRNA was evident in the center of NPBs in most blastomeres’nuclei of untreated control embryos, such signals were not observed in their treated counterparts. D) Blastocysts were treated or not with Flavopiridol for 1 hour and simultaneously were labeled with FU. Then embryos were fixed and co-immunostained against FU and CDK9. In control group, the transcriptional activity was at higher level in the areas with less DAPI staining and corresponding to nucleoli. CDK9 also predominated in these areas. In treated embryos, both nucleoplasmic and nucleolar nascent RNAs were declined. Also nucleolar localization of CDK9 was unraveled. Only individual cells from the whole blastocysts are shown.

Article Snippet: CDK9 Inhibitor II/CAN508 (sc-203326) was purchased from Santa Cruz and dissolved in DMSO to form 5 mM stock solution.

Techniques: Labeling, Cell Culture, Immunostaining, Inhibition, Control, Activity Assay, Staining

Journal: Cell Death & Disease

Article Title: Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

doi: 10.1038/cddis.2011.66

Figure Lengend Snippet: Wogonin is a naturally occurring inhibitor of CDK7 and CDK9. ( a ) Wogonin inhibits phosphorylation of the CTD of RNAPII at Ser 2 and Ser 5 sites. CEM cells were treated with different concentrations of wogonin for 2 h (left panel) or for different time periods with 50 μ M wogonin (right panel) and the cells were analyzed for the status of RNAPII phosphorylation by western blot analysis using antibodies specific for phosphorylated CTD of RNAPII Ser 2 and Ser 5 sites. One representative experiment of three is shown. ( b ) Wogonin does not inhibit phosphorylation of the retinoblastoma (RB) protein. CEM cells were treated with 50 μ M wogonin (Wogo) for 3 h. Cells were lysed and total RB was immunoprecipitated and phosphorylated RB was examined by western blot using phospho-specific antibodies as indicated (left panel). As a control, the same cell lysates were analyzed for the status of phosphorylation of RNAPII at the Ser 2 residue (right panel). Data are representative of three independent experiments. ( c ) Wogonin inhibits CDK7 and CDK9 kinase activity determined by incorporation of [ 33 P]. CDK7/cyclinH/MAT1 or CDK9/cyclinT was incubated with substrate peptide and [ 33 P]-ATP in the presence of different doses of wogonin as indicated. The kinase activity is described as % of [ 33 P]-phosphorylated substrate peptide. Means±S.D. are shown. The half-maximal inhibitory concentrations (IC 50 ) are indicated

Article Snippet: Specific knockdown of CDK9 expression in CEM cells by RNA interference resulted in induction of apoptosis to a similar extent as the knockdown of Mcl-1 ( ).

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Control, Residue, Activity Assay, Incubation

Journal: Cell Death & Disease

Article Title: Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

doi: 10.1038/cddis.2011.66

Figure Lengend Snippet: In silico docking analysis of wogonin on the crystal structure of CDK9. ( a ) Computer docking simulation of the crystal structure of human CDK9 in complex with wogonin. ( b ) MultiBind webserver analysis predicted wogonin-binding residues in CDK9. The residues involved in the binding of wogonin to CDK9 are indicated by triangles

Article Snippet: Specific knockdown of CDK9 expression in CEM cells by RNA interference resulted in induction of apoptosis to a similar extent as the knockdown of Mcl-1 ( ).

Techniques: In Silico, Binding Assay

Journal: Cell Death & Disease

Article Title: Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

doi: 10.1038/cddis.2011.66

Figure Lengend Snippet: Several natural flavones inhibit CDK9 function. ( a ) Chemical structures of apigenin, chrysin, luteolin and wogonin. ( b ) Apigenin, chrysin and luteolin inhibit phosphorylation of RNAPII and downregulate Mcl-1 expression. Leukemic CEM cells were treated with 50 μ M of different flavones for indicated time periods. Cell lysates were subjected to western blot analysis using antibodies as indicated. Results are representative of two independent experiments. ( c ) Apigenin, chrysin and luteolin induce apoptosis in leukemic cells. Leukemic CEM cells were treated with different concentrations of the indicated flavones for 24 h. Apoptotic cell death was determined by DNA fragmentation. Means±S.D. are shown. Results are representative of two independent experiments. ( d ) Flavones directly interact with CDK9. Upper panel, a flavone backbone coupled to Affi-Gel-10 and the Affi-Gel coupled to the linker only were used to analyze flavone-binding proteins. Lower panel left, leukemic CEM cells were treated with 50 μ M of flavone with linker (FL) or negative control (NC) for 24 and 48 h. Apoptotic cell death was determined by DNA fragmentation. Means±S.D. are shown. Lower panel right, western blot analysis of proteins pulled down from the flavone-coupled or the negative control-coupled Affi-Gel beads. HSP90 was used to control for equal unspecific binding. The result is representative of two independent pull-down experiments

Article Snippet: Specific knockdown of CDK9 expression in CEM cells by RNA interference resulted in induction of apoptosis to a similar extent as the knockdown of Mcl-1 ( ).

Techniques: Phospho-proteomics, Expressing, Western Blot, Binding Assay, Negative Control, Control

Journal: Cell Death & Disease

Article Title: Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

doi: 10.1038/cddis.2011.66

Figure Lengend Snippet: Inhibition of Mcl-1 or CDK9 expression is sufficient to mimic wogonin-induced apoptosis. ( a ) Knockdown of Mcl-1 induces apoptosis. HCT116 and CEM cells were transfected with either siRNA specific for Mcl-1 or scrambled siRNA as described in Materials and Methods. Knockdown efficiency was controlled by western blot at 48 h after transfection. Apoptotic cell death was determined by DNA fragmentation at 48 h and 72 h after transfection for HCT116 and CEM, respectively. Means±S.D. are shown. The result is representative of two independent knockdown experiments. ( b ) Overexpression of Mcl-1 inhibits wogonin-induced apoptosis. HCT116 cells were transfected with either the Mcl-1 expressing plasmid (pMcl-1) or the parental control plasmid (pEF4) as described in Materials and Methods. The overexpression efficiency was controlled by western blot at 24 h after transfection. Cells were treated with 50 μ M wogonin for 24, 48 and 72 h as indicated. Apoptotic cell death was determined by DNA fragmentation. Means±S.D. are shown. The result is representative of two independent experiments. ( c ) Knockdown of CDK9 is sufficient to induce apoptosis. CEM cells were transfected with either siRNA #1 or #2 specific for CDK9 or control siRNA. Knockdown efficiency was controlled by western blot at 24 or 48 h after transfection. Apoptotic cell death was determined by DNA fragmentation at 72 h after transfection. The result is representative of two independent knockdown experiments. Means±S.D. are shown. ( d ) Model showing the mechanism of the anti-tumor and anti-viral effect of flavones

Article Snippet: Specific knockdown of CDK9 expression in CEM cells by RNA interference resulted in induction of apoptosis to a similar extent as the knockdown of Mcl-1 ( ).

Techniques: Inhibition, Expressing, Knockdown, Transfection, Western Blot, Over Expression, Plasmid Preparation, Control

Journal: Cell Death & Disease

Article Title: Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

doi: 10.1038/cddis.2011.66

Figure Lengend Snippet: Comparative studies of the effects of wogonin on CDK9 activity in leukemic and normal T cells. ( a ) Wogonin induces apoptosis in malignant T cells and not in normal activated T cells. CEM and activated T cells isolated from peripheral blood of three representative healthy donors were treated with different concentrations of wogonin for 48 h. Apoptotic cell death was determined by DNA fragmentation. Means±S.D. are shown. ( b ) Malignant T cells have higher levels of phosphorylated RNAPII compared with normal activated T cells. Phosphorylation of RNAPII at Ser 2 in peripheral blood T cells from five healthy donors and different malignant T cell lines was analyzed using western blot. ( c ) Wogonin exerts stronger inhibitory effects on CDK9 in malignant than in normal T cells. Peripheral blood T cells from donors in a were treated with 50 μ M of wogonin for different time periods as indicated. The effects of wogonin on CDK9 activity were examined by western blot for phosphorylation of RNAPII at Ser 2 . CEM cells were treated in parallel as a control. Results are presented as amount of Ser 2 phosphorylated RNAPII relative to total RNAPIIa. ( d ) Representative western blot from donor 1 is shown

Article Snippet: Specific knockdown of CDK9 expression in CEM cells by RNA interference resulted in induction of apoptosis to a similar extent as the knockdown of Mcl-1 ( ).

Techniques: Activity Assay, Isolation, Phospho-proteomics, Western Blot, Control

Journal: Wiley Interdisciplinary Reviews. RNA

Article Title: Cyclin‐dependent kinases: Masters of the eukaryotic universe

doi: 10.1002/wrna.1816

Figure Lengend Snippet: Transcriptional CDKs in metazoans.

Article Snippet: Several other groups also showed that selective inhibition of CDK7 leads to cell cycle arrest, apoptosis, and decreased transcription levels, especially of genes associated with super‐enhancers (Galbraith et al., ; Sava et al., ).

Techniques: Binding Assay, Activity Assay

Journal: Chemistry & Biology

Article Title: Specific CLK Inhibitors from a Novel Chemotype for Regulation of Alternative Splicing

doi: 10.1016/j.chembiol.2010.11.009

Figure Lengend Snippet: Effect of the Studied Inhibitors on Enzymatic Activity

Article Snippet: K00546 is CDK1/2 inhibitor III purchased from Merck Biosciences (cat. # 217714).

Techniques:

Journal: Chemistry & Biology

Article Title: Specific CLK Inhibitors from a Novel Chemotype for Regulation of Alternative Splicing

doi: 10.1016/j.chembiol.2010.11.009

Figure Lengend Snippet: Data Collection and Refinement Statistics

Article Snippet: K00546 is CDK1/2 inhibitor III purchased from Merck Biosciences (cat. # 217714).

Techniques:

Journal: Chemistry & Biology

Article Title: Specific CLK Inhibitors from a Novel Chemotype for Regulation of Alternative Splicing

doi: 10.1016/j.chembiol.2010.11.009

Figure Lengend Snippet: Binding Mode of CLK Inhibitors (A) Overview of the CLK cocrystal structure. The CLK1 catalytic domain is shown as a ribbon diagram and the ATP binding site has been highlighted in surface representation. Details of the interaction of KH-CB19 with the kinase active site are shown in the detailed view on the right. (B) Superimposition of the CLK3 cocrystal structure with KH-CB19 and the triazole diamine K00546. (C) Superimposition of CLK1 complexes with KH-CB19 and hymenialdisine (K0010). (D) Electron density (2Fc-2Fo) map of KH-CB19 in the CLK1 complex. The map has been contoured using a 2 sigma cutoff.

Article Snippet: K00546 is CDK1/2 inhibitor III purchased from Merck Biosciences (cat. # 217714).

Techniques: Binding Assay