Journal: eLife

Article Title: The PMA phorbol ester tumor promoter increases canonical Wnt signaling via macropinocytosis

doi: 10.7554/eLife.89141

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , CD63-RFP ( Homo sapiens ) (plasmid) , Addgene , RRID: Addgene_62964 , RFP tag.

Techniques: Recombinant, Dominant Negative Mutation, Plasmid Preparation, Membrane, Luciferase, Expressing, Reporter Assay, Software, Imaging, Microinjection, Inverted Microscopy

Journal: bioRxiv

Article Title: Klebsiella pneumoniae reduces SUMOylation to limit host defence responses

doi: 10.1101/2020.06.29.179275

Figure Lengend Snippet: A. Immunoblot analysis of Ubc9 and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. Ubc9 bands were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to non-infected control cells. n.s. – non significant; versus n.i. determined using one way-ANOVA with Tukey’s multiple comparisons test. B. Immunoblot analysis of Sae1, Sae2 and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. Sae1 and Sae2 bands were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to non-infected control cells. n.s. – non significant; versus n.i. determined using one way-ANOVA with Tukey’s multiple comparisons test. C. Immunoblot analysis of SUMO1 and tubulin levels in lysates of control (AS), SENP1 or SENP2 siRNA-transfected A549 cells infected with Kp52145 for 5 h. AS-AllStars control, non-silencing siRNA. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to AS-transfected non-infected control cells. ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; versus AS n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. D.Fluorescence microscopy of pSENP2-GFP transfected A549 cells grown on glass coverslips. Cells were infected with Kp52145 for 5 h or left uninfected (n.i.). Coverslips were stained with Hoechst (DAPI) for nuclei identification. The % of SENP2 localised in either nucleus or cytoplasm is represented on the graph and is the result of independent counting of 100 cells from each of 3 independent experiments. E.Immunoblot analysis of SENP2 and tubulin levels in cytosolic extracts of A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). F.Fluorescence microscopy of pSENP2-GFP transfected A549 cells grown on glass coverslips. Cells were treated with the proteasomal inhibitor MG262 (5 μM, 2 h before infection), the nuclear export inhibitor Leptomycin B (LMB, 10 nM, 2 h before infection) or DMSO (vehicle solution) and infected with Kp52145 for 5 h or left uninfected (n.i.). Coverslips were stained with Hoechst (DAPI) for nuclei identification. The % of SENP2 localized in either nucleus or cytoplasm is represented on the graphs and is the result of independent counting of 100 cells from each of 3 independent experiments. G.Immunoblot analysis of SUMO1 and tubulin levels in lysates of the nuclear export inhibitor Leptomycin B (LMB, 5 μM, 2 h before infection) or DMSO (vehicle solution)-treated A549 infected with Kp52145 for 5 h or left uninfected (n.i.). SUMO1 smears were quantified using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to DMSO non-infected control cells. **P ≤ 0.01 versus DMSO n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. H.Immunoblot analysis of K48-linkage specific polyubiquitin and FLAG (DYKDDDDK-protein tag) levels in immunoprecipitates of A549 infected with Kp52145 for 5 h. Cells were immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. I.Immunoblot analysis of SENP2 and tubulin levels in cytosolic extracts of control (AS), Rbx1, Skp1, Cul-1, βTrCP or Skp2 siRNA-transfected A549 cells left uninfected (n.i.). J.Immunoblot analysis of Cullin-1 and tubulin levels in lysates of A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). Lysates of A549 treated with 5 mM of H 2 O 2 for 10 min were used as a positive control for Cullin-1 deNEDDylation. K.Immunoblot analysis of Cullin-1 and tubulin levels in lysates of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. L.Immunoblot analysis of K48-linkage specific polyubiquitin and FLAG (DYKDDDDK-protein tag) levels in immunoprecipitates of control (AS) or CSN5 siRNA-transfected A549. Cells were transfected with a SENP2-FLAG plasmid 24 h after the siRNA transfection and infected the following day with Kp52145 for 5 h or left uninfected. Cells were immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. M.Immunoblot analysis of Senp2 and tubulin levels in cytosolic extracts of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. N.Immunoblot analysis of SUMO1 and tubulin levels in lysates of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected (n.i.). AS-AllStars control, non-silencing siRNA. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (Li-cor) and the modification normalised to α-Tubulin. The graph represents fold change compared to AS-transfected non-infected control cells. **P ≤ 0.01 versus AS n.i. determined using one way-ANOVA with Bonferroni’s multiple comparisons test. In all panels data are representative of at least three independent experiments.

Article Snippet: Cells were transfected using Lipofectamine 2000 with plasmids encoding pEGFP-C2 SENP2 (gift from Mary Dasso, Addgene plasmid # 13382) and infected 24 h later.

Techniques: Western Blot, Infection, Modification, Control, Transfection, Fluorescence, Microscopy, Staining, Immunoprecipitation, Negative Control, Positive Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Klebsiella pneumoniae reduces SUMOylation to limit host defence responses

doi: 10.1101/2020.06.29.179275

Figure Lengend Snippet: A. ELISA of IL-8 secreted by A549 cells transfected with either empty control plasmid (pcDNA3) or SUMO1-HA tagged plasmid (pSUMO1), which were left untreated (n.i.) or infected with Kp52145 for 3 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. B. ELISA of IL-8 secreted by control (AS) or Senp2 siRNA-transfected A549, which were left untreated (n.i.) or infected with Kp52145 for 3 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus AS determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. C.ELISA of TNFα secreted by empty control (pcDNA3) or SUMO1-HA plasmid-transfected MH-S, which were left untreated (n.i.) or infected with Kp52145 for 1 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. D.ELISA of TNFα secreted by antagomir-transfected MH-S, which were left untreated (n.i.) or infected with Kp52145 for 1 h of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus Negative Kp52145 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. E.Immunoblot analysis of IκBα and tubulin levels in lysates of empty (pcDNA3) or SUMO1-HA (pSUMO1) plasmid transfected A549 or MH-S cells, infected with Kp52145 for the indicated time points. F.Immunoblot analysis of JNK (P-JNK), ERK (P-ERK) and p38 (P-p38) phosphorylation levels in lysates of empty (pcDNA3) or SUMO1-HA (pSUMO1) plasmid transfected A549 or MH-S cells, infected with Kp52145 for the indicated time points. Tubulin immunoblotting was used as the loading control. n.i. – non-infected control G.ELISA of IL-8 or TNFα secreted by empty control (pcDNA3) or SUMO1-HA plasmid-transfected A549 or MH-S respectively, which were treated with the NF-κB inhibitor BAY 11-7082 (5 μM, 2 h before infection) or DMSO (vehicle solution). Cells were left untreated (n.i.) or infected with Kp52145 for 1 h (MH-S) or 3 h (A549) of contact after which the medium was replaced with medium containing gentamicin (100 µg mL -1 ) to kill extracellular bacteria and incubated for a further 4 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. H.Percentage of intracellular survival in MH-S cells transfected with either empty control plasmid (pcDNA3) or SUMO1-HA tagged plasmid (pSUMO1). Cells were infected with Kp52145 for 30 min (MOI 100:1), wells were washed and incubated with medium containing gentamicin (100 μg mL -1 ) for 2.5 h. Intracellular bacteria were quantified by lysis, serial dilution and viable counting on LB agar plates. Percentage of intracellular survival was determined by dividing the number of colony forming units (CFU) obtained at 3 h of infection over the number obtained at 1 h and considering pcDNA3 as 100 %. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using unpaired t-test with correction for Holm-Sidak’s multiple comparisons test. I.Percentage of intracellular survival in antagomir transfected MH-S cells. Cells were infected with Kp52145 for 30 min (MOI 100:1), wells were washed and incubated with medium containing gentamicin (100 μg mL -1 ) for 2.5 h. Intracellular bacteria were quantified by lysis, serial dilution and viable counting on LB agar plates. Percentage of intracellular survival was determined by dividing the number of colony forming units (CFU) obtained at 3 h of infection over the number obtained at 1 h and considering the negative control as 100 %. Values are presented as the mean ± SD of three independent experiments measured in duplicate. **P ≤ 0.01; *P ≤ 0.05; versus negative control determined using one way-ANOVA with Holm-Sidak’s multiple comparisons test. In panels E and F, data are representative of at least three independent experiments.

Article Snippet: Cells were transfected using Lipofectamine 2000 with plasmids encoding pEGFP-C2 SENP2 (gift from Mary Dasso, Addgene plasmid # 13382) and infected 24 h later.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Control, Plasmid Preparation, Infection, Bacteria, Incubation, Western Blot, Phospho-proteomics, Lysis, Serial Dilution, Negative Control

Journal: bioRxiv

Article Title: Klebsiella pneumoniae reduces SUMOylation to limit host defence responses

doi: 10.1101/2020.06.29.179275

Figure Lengend Snippet: Working model of K. pneumoniae strategies to decrease SUMOylation in epithelial cells and macrophages. In epithelial cells, Kp52145 activates the signalling pathway EGFR-PI3K-AKT-ERK-GSK3β to increase the COP9 signalosome component CSN5 which inhibits NEDDylation of Cullin-1 and prevents proteasomal degradation of SENP2. SENP2 then accumulates in the cytosol preventing SUMOylation. In macrophages, Kp52145 via TLR4-TRAM-TRIF induces the production of type-I interferon, which signals through the IFNAR1 receptor. Type-I interferon stimulates transcription of the miRNA let-7 which prevents SUMOylation. Both strategies lead to increased intracellular survival and subversion of host responses.

Article Snippet: Cells were transfected using Lipofectamine 2000 with plasmids encoding pEGFP-C2 SENP2 (gift from Mary Dasso, Addgene plasmid # 13382) and infected 24 h later.

Techniques:

Journal: Oncogene

Article Title: MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage and instability

doi: 10.1038/onc.2017.193

Figure Lengend Snippet: miR-20a targets several autophagy related genes. ( a ) Relative mRNA levels (normalized by GAPDH) of BECN1, SQSTM1, ATG16L1 in MCF7 or ( c ) MDA-MB-231 cells overexpressing NC or miR-20a were analyzed by qPCR. ( b ) MCF7 or ( d ) MDA-MB-231 cells transfected with NC, miR-20a, LNA-NC or LNA-20a were either untreated or treated with EBSS for 4 h. The protein levels of ATG16L1, BECN1 and SQSTM1 were analyzed by immunoblotting. ( e ) Schematic illustration of the predicted miR-20a pairing sites in BECN1, SQSTM1 and ATG16L1 3′UTRs. Luciferase reporters containing wild type (WT) or mutated (MUT) BECN1, SQSTM1 or ATG16L1 3′UTR fragments were co-transfected with NC or miR-20a into MCF7 cells. Relative luciferase activity was normalized to NC (** P <0.01).

Article Snippet: SQSTM1 plasmid was purchased from Addgene (Cambridge, MA, USA; 28027); BECN1-GFP and FLAG-ATG16L1 plasmids were purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Transfection, Western Blot, Luciferase, Activity Assay

Journal: Oncogene

Article Title: MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage and instability

doi: 10.1038/onc.2017.193

Figure Lengend Snippet: miR-20a increases intracellular ROS levels and DNA damage. ( a ) Immunofluorescence of γH2AX in MCF7 cells overexpressing NC or miR-20a, γH2AX fluorescence intensity was quantified with Image J software (*** P <0.001). ( b ) Protein expression of γH2AX and GAPDH were detected by immunoblotting in MCF7 cells overexpressing miRNAs or LNA inhibitors. ( c ) Comet assay in MCF7 cells overexpressing NC or miR-20a. Tail moment (TM) of at least 50 cells in three independent experiments were analyzed by the specific software CASP (** P <0.01). ( d ) MCF7 cells overexpressing miRNAs or siRNAs were cultured in normal medium or EBSS medium for 4 h. Intracellular ROS levels were determined by Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit. ( e ) The expression levels of γH2AX, BECN1, SQSTM1, ATG16L1, LC3 and ACTB in MCF7 cells transfected with siRNAs were determined by western blotting. ( f ) Relative γH2AX protein expression (normalized by ACTB) was determined by Image J densitometric analysis. ( g ) DQ Red BSA fluorescence intensity in MCF7 cells transfected with siRNAs against ATG16L1, BECN1 or SQSTM1 (* P <0.05, ** P <0.01). ( h ) miR-20a and plasmids (BECN1-GFP, FLAG-ATG16L1, HA-SQSTM1) were co-transfected into MCF7 cells, cells were cultured in normal medium or EBSS medium for 4 h, samples were collected for immunoblotting analysis. ( i ) Relative γH2AX protein expression (normalized by ACTB) was determined by Image J densitometric analysis. ( j ) Proteolytic activity was analyzed in MCF7 cells expressing miR-20a and plasmids (* P <0.05).

Article Snippet: SQSTM1 plasmid was purchased from Addgene (Cambridge, MA, USA; 28027); BECN1-GFP and FLAG-ATG16L1 plasmids were purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Immunofluorescence, Fluorescence, Software, Expressing, Western Blot, Single Cell Gel Electrophoresis, Cell Culture, Transfection, Activity Assay

Journal: Oncogene

Article Title: MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage and instability

doi: 10.1038/onc.2017.193

Figure Lengend Snippet: Expression of miR-20a and its target genes in breast cancer specimens. ( a ) Expression of miR-20a in normal breast ( n =30) and breast tumor tissues ( n =30) was determined by in situ hybridization. ( b ) miRNA in situ hybridization and HE staining in tissues from a triple-negative breast cancer patient. ( c ) Immunohistochemical analysis of γH2AX, BECN1, SQSTM1 and ATG16L1 in triple-negative breast cancer tissues and adjacent normal tissues. ( d ) Immunoblotting analysis of SQSTM1, ATG16L1, BECN1 and OPTN in normal mammary tissues and triple-negative breast cancer tissues. Densitometric ratios of BCEN1, SQSTM1, OPTN and ATG16L1 versus ACTB were quantified by Image J software.

Article Snippet: SQSTM1 plasmid was purchased from Addgene (Cambridge, MA, USA; 28027); BECN1-GFP and FLAG-ATG16L1 plasmids were purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Expressing, In Situ Hybridization, Staining, Immunohistochemical staining, Western Blot, Software

Journal: Oncogene

Article Title: MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage and instability

doi: 10.1038/onc.2017.193

Figure Lengend Snippet: Higher expression of miR-20a is associated with downregulation of BECN1 , ATG16L1 and SQSTM1 . ( a ) Correlation between miR-20a and BECN1, SQSTM1, and ATG16L1 was determined using Spearman coefficient analysis in TCGA breast cancer samples ( n =500). ( b ) The expression of BECN1, SQSTM1 and ATG16L1 in triple-negative breast cancers ( n =82) and other subtypes ( n =391).

Article Snippet: SQSTM1 plasmid was purchased from Addgene (Cambridge, MA, USA; 28027); BECN1-GFP and FLAG-ATG16L1 plasmids were purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Expressing

Journal: Oncogene

Article Title: MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage and instability

doi: 10.1038/onc.2017.193

Figure Lengend Snippet: miR-20a promotes tumorigenesis in a xenograft mouse model. ( a ) Genomic instability (fraction of copy-number altered genome and mutation counts) was detected in TCGA breast cancer patients ( P <0.001, n =347). ( b ) MDA-MB-231 cells stably expressing NC or miR-20a were injected into nude mice. miR-20a promotes tumorigenesis compared with NC at day 8 of injection. ( c ) miR-20a promotes tumor growth in vivo . Tumor growth curve was determined by measuring the width and length every day (* P <0.05, ** P <0.01, *** P <0.001). ( d ) Oncogenic miR-20a directly targets ATG16L1, BECN1 and SQSTM1, inhibits autophagic flux and lysosomal proteolytic activity, increases ROS levels and accumulates DNA damage, which likely contribute to genome instability and tumor-initiating capacity.

Article Snippet: SQSTM1 plasmid was purchased from Addgene (Cambridge, MA, USA; 28027); BECN1-GFP and FLAG-ATG16L1 plasmids were purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Mutagenesis, Stable Transfection, Expressing, Injection, In Vivo, Activity Assay

Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 2. Constitutively active MPF abolishes the G2 phase arrest caused by HuCdc6 overexpression. G2 phase HeLa cells were microinjected with (A) pEGFP-HuCdc6, pCyclin B1 and a constitutively active pCDK1AF or (B) with pEGFP-HuCdc6, pCyclin B1 and pCDK1wt. As controls we co-injected pCyclin B1 and pCDK1AF or pCyclin B1 and pCDK1wt were injected without HuCdc6 as a control (C). Approximately 100 fluorescent cells were counted for each sample. Fluorescence and DIC images were taken 7 h after injection. Arrows show mitotic cells. (C) The numbers of cells in G2 phase, mitosis and in early G1 phase were scored and calculated as a percentage of the total of injected cells. At least three independent experiments were performed and representative images are shown.

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: Over Expression, Injection, Control, Fluorescence

Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 1. In vivo analysis of HuCdc6 overexpression in G2 cells. (A) pEGFP and (B) pEGFP-HuCdc6 were microinjected into the nucleus of G2 phase HeLa cells. The behaviour of injected cells was observed by time-lapse fluorescence and DIC microscopy and images were taken every 30 min, or every 3 min after entry into mitosis over a 10 h period. Approximately 100 fluorescent cells expressing GFP or HuCdc6–GFP were classified according to their cell cycle stage: G2 phase, mitosis and after completion of mitosis (early G1 phase) during different time points starting from 1 h after microinjection. These numbers were compared with the total number of cells expressing GFP or HuCdc6–GFP (C and D). While 40–45% of cells expressing GFP went through mitosis [arrows in (A) and (B) show mitotic cells], only 3–5% of cells expressing HuCdc6–GFP did so. Representative images, mean values and standard deviations of five independent experiments are shown. (E) Comparison of expression of HuCdc6–GFP to endogenous HuCdc6 levels. A total of 1000 cells were injected with pEGFP-HuCdc6 and after 2 h were directly lysed in SDS buffer, proteins were separated on 10% SDS–PAGE and immunoblotted for HuCdc6. (F) Anti human Cdc6 antibody neutralizes HuCdc6 overexpression and abolishes the G2 phase arrest. G2 phase HeLa cells were microinjected with HuCdc6–GFP and a specific anti HuCdc6 antibody. As a control, cells were injected with the anti HuCdc6 antibody and Texas Red dextran. The percentages of cells expressing pEGFP-HuCdc6 in G2, M and G1 phases were calculated and compared with the control cells 7 h after injection. Mean values and standard deviation were calculated from at least three independent experiments.

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: In Vivo, Over Expression, Injection, Fluorescence, Microscopy, Expressing, Microinjection, Comparison, SDS Page, Control, Standard Deviation

Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 3. Overexpression of Cdc25 abolishes the G2 phase arrest caused by HuCdc6–GFP. G2 phase cells were microinjected with pCdc25B and pEGFP-HuCdc6 (A) or with pEGFP-Cdc25C and pHuCdc6 (B). Arrows in (A) and (B) show mitotic cells. Cells were followed by time-lapse fluorescence and DIC microscopy as described in Figure 1, starting from 2 h after microinjection during a 7 h time course. Sixty per cent of cells entered mitosis prematurely and arrested in mitosis whether they expressed HuCdc6–GFP and Cdc25B (C) or Cdc25B alone (E). (D) Forty five per cent of cells expressing Cdc25C and HuCdc6–GFP entered and progressed through mitosis, but often with a 4 h delay compared with the 2 h delay of the control cells expressing Cdc25C only (F).

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: Over Expression, Fluorescence, Microscopy, Microinjection, Expressing, Control

Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 4. An inhibitor of Chk1 kinase activity, UCN-01, abolishes HuCdc6-mediated G2 arrest. (A) G2 phase HeLa cells were microinjected with pEGFP-HuCdc6 or pEGFP as a control (not shown). UCN-01 (300 nM) was added to the medium immediately after the injections. Fluorescence and DIC images were taken during a 10 h period. G2 phase and mitotic cells (marked with an arrow) were counted from a total of ∼100 fluorescent cells over a 10 h period. (B) Percentage of cells progressing through mitosis expressing HuCdc6 or GFP. Twenty-one percent of cells expressing HuCdc6–GFP entered and progressed through mitosis comparable with the 24% of GFP expressing cells. Two experiments were performed and representative images are shown. (C) HuCdc6-mediated phosphorylation of Chk1. One thousand cells were injected with pEGFPHuCdc6 or pEGFP, uninjected but untreated or treated with HU (control cells). Proteins were separated on SDS–PAGE and immunoblotted for Chk1. Lane 1 shows a mobility shift due to phosphorylation after HU treatment, whereas control cells do not (lanes 2, 4 and 6). HuCdc6 shows a similar mobility shift as HU-treated cells (lane 1), whereas GFP alone does not (lane 3). (D) The HuCdc6-mediated G2 phase arrest is maintained in the presence of caffeine and/or wortmannin. Cells were microinjected with pEGFP-HuCdc6 in the presence or absence of 5 mM caffeine and/or 25 µM wortmannin. As a control, cells were γ-irradiated (100 kVp/min) for 15 min and caffeine and/or wortmannin were added. Cells were followed for 10 h counting cells entering and progressing through mitosis as one (%M + G1). Uninjected cells and irradiated cells entered into mitosis prematurely in the presence of caffeine and/or wortmannin. The cells expressing HuCdc6 remained arrested in G2 phase in the presence of caffeine and/or wortmannin.

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: Activity Assay, Control, Fluorescence, Expressing, Phospho-proteomics, Injection, SDS Page, Mobility Shift, Irradiation

Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 5. Behaviour of HuCdc6 mutants in G2 cells. (A) Schematic drawing of HuCdc6 depicting features such as the CDK phosphorylation sites serines (S) 54, 74 and 106, destruction box, KEN box, the cyclin-binding-motif, the ATPase/ORC homology domain and leucine-zipper, all previously identified. (B) The pEGFP-HuCdc6 mutants S54A, S74A, S106A and Δcy-motif were injected into G2 HeLa cells and their progression into mitosis compared with GFP or wtHuCdc6–GFP-expressing cells. Cells expressing the mutants S75A and Δcy-motif cannot arrest cells in G2, whereas cells expressing S54A and S106A do arrest cells in G2 in the same fashion to wtHuCdc6. (C) Illustration of the localization of the mutants. Two independent experiments were performed and representative images are shown.

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: Phospho-proteomics, Binding Assay, Injection, Expressing

Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 6. Model for potential checkpoint function of HuCdc6. Ongoing DNA replication is monitored either by proteins at the replication fork, including ATR, or by soluble factors, including HuCdc6. ATR and HuCdc6 might work in parallel pathways, both leading to the activation of Chk1 and consequent inactivation of Cdc25. The major role of the ATR pathway would be in response to stalled replication forks. Chk1 may also increase the activity of Wee1 that further prevents the activation of MPF.

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: Activation Assay, Activity Assay