Journal: Cancer Research

Article Title: CIP2A Modulates Cell-Cycle Progression in Human Cancer Cells by Regulating the Stability and Activity of Plk1

doi: 10.1158/0008-5472.can-13-0888

Figure Lengend Snippet: Figure 1. CIP2A is a cell-cycle regulated protein. A, HeLa cells were synchronized by either a double thymidine block or nocodazole block, released into fresh medium at indicated time points, and then analyzed by immunoblotting with antibodies against the indicated proteins. Synchronization and progression through the cell cycle was confirmed by fluorescence-activated cell sorting analysis. B, HeLa cells were stained with anti-CIP2A antibody (green), anti-pericentrin antibody (red), and DAPI (DNA, blue). C, an enlarged single-cell image from HeLa cells stained with anti-CIP2A antibody (green) and DAPI (blue). D, H1299 cells were transfected with the PTEN-expressing construct or empty vector. E, Hs68 cells were transfected with either control (Ctrl) siRNA or Plk1 siRNA. D and E, after transfection for 48 hours, PTEN-induced G1 arrest or Plk1 depletion–induced G2–M arrest was analyzed by immunoblotting with antibodies against the indicated proteins or by fluorescence-activated cell sorting analysis, respectively. F, Hs68 cells were stained with anti-CIP2A antibody (green) and anti–phospho-H3 antibody (red) or anti-cyclin B1 antibody (red) with DAPI (blue). Data shown represent typical results from at least four independent experiments. Scale bars, 10 mm.

Article Snippet: Paraformaldehyde-fixed HeLa cells were permeabilized with 0.2% Triton X-100, washed, and blocked with blocking solution (Olink Bioscience).

Techniques: Blocking Assay, Western Blot, FACS, Staining, Transfection, Expressing, Construct, Plasmid Preparation, Control

Journal: Cancer Research

Article Title: CIP2A Modulates Cell-Cycle Progression in Human Cancer Cells by Regulating the Stability and Activity of Plk1

doi: 10.1158/0008-5472.can-13-0888

Figure Lengend Snippet: Figure 2. CIP2A depletion blocks nocodazole-induced mitotic arrest. A, HeLa cells were transfected with the indicated siRNAs (top) or transfected with control (Ctrl) siRNA, CIP2A.1 siRNA, or both CIP2A.1 siRNA and CIP2Arv (bottom) for 48 hours and then analyzed by immunoblotting with anti-CIP2A antibody. B, C, F, and G, HeLa cells were transfected with the indicated siRNAs or Flag-CHFR vector for 36 hours and then incubated with 100 ng/mL nocodazole for 16 hours. MAD2 siRNA or the Flag-CHFR vector was used for a positive control of spindle checkpoint regulation or premitotic regulation, respectively. Cells were analyzed by light microscopy (B, left), fluorescence-activated cell sorting analysis (B, right), or immunoblotted with antibodies against the indicated proteins (C). D and E, HeLa cells were transfected with control (Ctrl) siRNA, CIP2A.1 siRNA, or both CIP2A.1 siRNA and CIP2Arv for 48 hours and then incubated with 100 ng/mL nocodazole for 16 hours. Nocodazole-treated cells were stained with anti-CIP2A antibody (green) and anti–phospho-H3 antibody (red) with DAPI (blue; D). E, the mitotic index was determined by the percentage of phospho-H3–positive cells (bottom) or by FACS (top) and was quantified using CellProfiler software (300 cells for each data point, n ¼ 3; , P < 0.001). F, nocodazole-treated cells were fixed with DAPI (blue) and the percentage of nonmitotic cells with multilobed or interphase nuclei was quantified (300 cells for each data point, n ¼ 3; , P < 0.01). G, representative confocal images of the cells that indicate the different nuclear morphologies, including mitotic arrest, checkpoint bypass, or premitotic arrest. Arrows, multilobed nuclei. Data shown represent typical results from at least three independent experiments. Scale bars, 20 mm. p-H3–positive, phospho-H3–positive.

Article Snippet: Paraformaldehyde-fixed HeLa cells were permeabilized with 0.2% Triton X-100, washed, and blocked with blocking solution (Olink Bioscience).

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Incubation, Positive Control, Light Microscopy, FACS, Staining, Software

Journal: Cancer Research

Article Title: CIP2A Modulates Cell-Cycle Progression in Human Cancer Cells by Regulating the Stability and Activity of Plk1

doi: 10.1158/0008-5472.can-13-0888

Figure Lengend Snippet: Figure 3. CIP2A depletion results in delay of mitotic entry and mitotic abnormalities. A, schematic of cell synchronization protocol by double thymidine block and for transfection with siRNA (top). A and B, HeLa cells were transfected with control (Ctrl) siRNA or CIP2A.1 siRNA, synchronized at the G1–S phase by a double thymidine block and released from the secondary thymidine block into medium with or without 100 ng/mL nocodazole (treated 6 hours after release). A, the mitotic index of control- or CIP2A-depleted cells was expressed as the percentage of phospho- H3–positive cells (500 cells at each time point; bottom). B, cells were analyzed by fluorescence-activated cell sorting (FACS) analysis (top) or immunoblotted with the indicated antibodies (bottom). C and D, control (Ctrl) or CIP2A shRNA knockdown cells expressing FUCCI probes were synchronized by double thymidine block and released with fresh medium. C, the time of mitotic entry was determined by observing mitotic cell rounding with signs of DNA condensation and was monitored by time-lapse microscopy (left). Stable knockdown of CIP2A was determined by immunoblotting with anti-CIP2A antibody (right). D, representative time-lapse images of control (Ctrl) or CIP2A shRNA knockdown cells expressing FUCCI probes during cell-cycle progression. E–H, HeLa cells were transfected with either control (Ctrl) siRNA or CIP2A.1 siRNA for 72 hours. E, CIP2Arv was cotransfected with CIP2A.1 siRNA for the rescue of CIP2A. The percentage of normal and aberrant nuclei was quantified using fluorescence microscopy (500 cells for each data point). F, representative images of CIP2A-depleted cells stained with anti-CIP2A antibody (green) and DAPI (blue). Arrows indicate aberrant nuclei. G, CIP2A-depleted cells were scored for abnormal mitosis (200 mitotic cells for each data point, n ¼ 4, error bars, SD). H, representative images of a normal mitotic cells (i) and CIP2A-depleted mitotic cells (ii–vi) stained with anti-CIP2A antibody (green), anti-pericentrin antibody (red), and DAPI (blue). The data shown represent typical results from at least three independent experiments. Scale bars, 10 mm. , P < 0.001. p-H3–positive, phospho-H3–positive; Noc, nocodazole.

Article Snippet: Paraformaldehyde-fixed HeLa cells were permeabilized with 0.2% Triton X-100, washed, and blocked with blocking solution (Olink Bioscience).

Techniques: Blocking Assay, Transfection, Control, FACS, shRNA, Knockdown, Expressing, Time-lapse Microscopy, Western Blot, Microscopy, Staining

Journal: Cancer Research

Article Title: CIP2A Modulates Cell-Cycle Progression in Human Cancer Cells by Regulating the Stability and Activity of Plk1

doi: 10.1158/0008-5472.can-13-0888

Figure Lengend Snippet: Figure 4. CIP2A binds to Plk1 during mitosis. A and B, HeLa cells were transfected with control (Ctrl) siRNA, CIP2A.1 siRNA, or both CIP2A.1 siRNA and Flag-Plk1 vector for 48 hours and then incubated with 100 ng/mL nocodazole for 16 hours. Cells were analyzed by light microscopy (A) or immunoblotted with antibodies against the indicated proteins (B). C, lysates of HeLa cells were immunoprecipitated with anti-CIP2A antibody, anti-Plk1 antibody, or their respective control immunoglobulin G (IgG) antibodies and immunoblotted with anti-CIP2A or anti-Plk1 antibody. D, lysates of HeLa cells released from thymidine block for the indicated times were immunoprecipitated with anti-CIP2A antibody and immunoblotted with anti-Plk1 or anti-CIP2A antibody. E, lysates of 293T cells expressing Flag, Flag-tagged Plk1 (WT), polo-box–mutant (FAA) Plk1, N-terminal (N) Plk1, and C-terminal (C) Plk1 with Strep-CIP2A were pulled down with Strep-Tactin beads. The Flag-Plk1 protein associated with Strep-CIP2A was detected by immunoblotting with anti-Flag antibody. F–I, HeLa cells were fixed and incubated with mouse anti-CIP2A antibody together with rabbit anti-phospho-Plk1 (Thr210) antibody (top) or rabbit anti-CIP2A antibody together with mouse anti-Plk1 antibody (bottom), followed by in situ PLA analysis. Arrows indicate mitotic cells (F). G and I, dots per cell were counted using CellProfiler (100 cells for each data point; G) or 20 single cells in each respective cell-cycle phase (I); error bars, SD, , P < 0.001). The variation in the number of signal dots between G and I was due to the size difference between multiple- and single-cell images. H, representative confocal images of cells with PLA-positive signals in each respective cell-cycle phase. Data shown represent typical results from at least three independent experiments. Scale bars, 20 mm. p-MPM2, phospho-MPM2.

Article Snippet: Paraformaldehyde-fixed HeLa cells were permeabilized with 0.2% Triton X-100, washed, and blocked with blocking solution (Olink Bioscience).

Techniques: Transfection, Control, Plasmid Preparation, Incubation, Light Microscopy, Immunoprecipitation, Blocking Assay, Expressing, Mutagenesis, Western Blot, In Situ

Journal: Cancer Research

Article Title: CIP2A Modulates Cell-Cycle Progression in Human Cancer Cells by Regulating the Stability and Activity of Plk1

doi: 10.1158/0008-5472.can-13-0888

Figure Lengend Snippet: Figure 5. CIP2A regulates the stability and activity of Plk1 during mitosis. A, HeLa cells were transfected with the indicated siRNAs, synchronized, and treated with cycloheximide (CHX), which was added 7 hours after release from G1–S. At the indicated times after the addition of cycloheximide, cells were analyzed by immunoblotting with the indicated antibodies. B, the levels of Plk1 were quantified using ImageJ software (n ¼ 3; error bars, SD, , P < 0.01). C, at 7 hours after release from G1–S, cells were treated with MG132 for 3 hours. D, cells cotransfected with vectors for HA-ubiquitin (Ub) and empty or CIP2A-Myc were released from G1–S for 1 hour (interphase) or 7 hours (mitosis) and MG132 was added for 3 hours. Lysates of the cells were immunoprecipitated with anti- Plk1 antibody and immunoblotted with anti-HA antibody. E, HeLa cells transfected with the indicated siRNAs targeting CIP2A, Cdh1, or Cdc20 were synchronized at the G1–S phase, released for 10 hours and then analyzed by immunoblotting with the indicated antibodies. F, HeLa cells transfected with control (Ctrl) siRNA or CIP2A.1 siRNA were synchronized by a double thymidine block and released into fresh medium. Cells were analyzed by immunoblotting with the indicated antibodies. G, purified Strep-CIP2A protein (1mg) incubated with or without recombinant His-Plk1 (0.1 mg) was used as thecontrol to exclude the possibility that purified Strep-CIP2A protein was associated with kinases that can phosphorylate casein (left). Recombinant His-Plk1 (0.1 mg) was incubated with 50 mmol/L BI2536 alone or with serial concentrations of purified Strep-CIP2A proteins (0.25, 0.5, or 1 mg) for 30 minutes (right). Plk1 activity was determined by in vitro kinase assay using casein as a substrate. Data shown represent typical results from at least three independent experiments.

Article Snippet: Paraformaldehyde-fixed HeLa cells were permeabilized with 0.2% Triton X-100, washed, and blocked with blocking solution (Olink Bioscience).

Techniques: Activity Assay, Transfection, Western Blot, Software, Ubiquitin Proteomics, Immunoprecipitation, Control, Blocking Assay, Incubation, Recombinant, In Vitro, Kinase Assay