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: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

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: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

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: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

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: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

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: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

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

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 6. Association between CIP2A and Plk1 in lung, gastric, colon, and cervical cancers and their normal tissue counterparts. A, quantification of CIP2A and Plk1 staining intensities in lung, gastric, colon, and cervical cancers and their normal tissue counterparts. B and C, representative microscopic images of lung, gastric, colon, and cervical cancers and their normal tissue counterparts stained with anti-CIP2A antibody (B) or anti-Plk1 antibody (C). Scale bars, 200 mm. D, representative high-magnification images of gastric cancer tissues stained with the indicated antibodies. Scale bars, 100 mm. E and F, tissue sections were incubated with rabbit anti-CIP2A antibody together with mouse anti-Plk1 antibody followed by in situ PLA analysis. Representative confocal images of lung, gastric, colon, and cervical cancers and their normal tissue counterparts (E, left and F). Scale bars, 20 mm. Four different areas were obtained for each sample and 200 to 500 cells were quantified per area (mm2) using CellProfiler (E, right). Nuclei were stained with Hoechst (blue). The green signal represents autofluorescence. N, normal lung tissue. C, cancer tissue. A and E (left), data are presented as box-and-whisker plots. , P < 0.001 compared with their normal tissue counterparts.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Staining, Incubation, In Situ, Whisker Assay

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

doi: 10.1038/s41467-022-31865-5

Figure Lengend Snippet: a Schematic showing the layout of conserved domains and regions in TOPBP1. Key amino acids in the AAD (W1145) and BRCT7 (K1317) are indicated. Deletion constructs of TOPBP1 used in b , lacking either the C-terminal portion of the protein (ΔC), the region between BRCT5 and 6 (Δ751-899) and BRCT domain 6 (ΔBRCT6). b Localization of GFP-TOPBP1 wild type and mutants in mitotic cells after 1 Gy of IR and interphase cells after 3 Gy of IR. Displayed are maximum intensity projections of confocal z-stacks. All scalebars = 10 µm. c HA-immunoprecipitation from 293FT cells transfected with a HA-tagged TOPBP1 fragment spanning the entire region between BRCT5 and 6 (amino acids 740-899). d HA-immuno-precipitation of the TOPBP1 fragment in c with the purified GST-ArmRP of CIP2A. e Flag-immunoprecipitation from 293FT transfected with Flag-tagged full-length CIP2A and treated with 3 Gy of IR and Nocodazole as indicated. f Quantification of the experiment in e . Columns represent the mean ratio between TOPBP1 and Flag band intensities in the Flag-IP blots, error bars represent the SEM of four independent experiments. Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: GFP (Mouse, Roche, 11814460001, 1:5000), GFP (Rabbit, Abcam, ab290, 1:1000), HA (Rabbit, Abcam, ab9110, 1:4000), MDC1 (Rabbit, Abcam, ab11171, 1:5000), MDC1 pSer168 (Rabbit, AMS Biotechnology, custom-made, 1:500) , MDC1 pSer196 (Rabbit, 21 st Century, custom-made 1:200) , TOPBP1 (Rabbit, Abcam, ab2402, 1:1500), CIP2A (Rabbit, Cell Signaling, 14805, 1:1000), CIP2A (Mouse, Santa Cruz, sc-80659, 1:1000), CRM1 (Rabbit, Atlas Antibodies, HPA042933, 1:1500), Tubulin (Mouse, Sigma, T6199, 1:5000), Tubulin hFABTM rhodamine (Bio-Rad, 12004166, 1:5000), FLAG M2 (Mouse, Sigma, F1804, 1:1000), pSer10 Histone H3 (Rabbit, Millipore, 06-570, 1:1000), Histone H3 (Rabbit, Abcam, ab1791, 1:10000).

Techniques: Construct, Immunoprecipitation, Transfection, Purification

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

doi: 10.1038/s41467-022-31865-5

Figure Lengend Snippet: a Confocal micrograph (maximum intensity projection) of untreated U2OS cells, stained for TOPBP1 and CIP2A. Centrosomes are highlighted with white arrowheads. b Confocal micrograph (maximum intensity projection) of Nocodazole-arrested U2OS cells 1 h after irradiation with 1 Gy, stained for TOPBP1 and CIP2A. c Upper panels: confocal micrographs of interphase U2OS cells treated with 3 Gy and U2OS cells arrested in mitosis by Nocodazole and treated with 1 Gy. Lower panels: micrographs deconvoluted and segmented by SQUASSH d Quantitative analysis of CIP2A and TOPBP1 colocalization by SQUASSH. Left: object size colocalization (area of object overlap divided by total object area). Right: object number colocalization (fraction of objects in each channel that overlap ≥ 50%). Each data point represents one cell ( n = 37; pooled from three independent experiments). Bars and error bars represent mean and SD. Statistical significance was assessed by two-sided unpaired t-tests (α = 0.05) e Airyscan high-resolution confocal image (maximum intensity projection) of CIP2A and TOPBP1 foci in mitosis 1 h after 1 Gy of IR. Scale bar in the merge panel: 5 µm; scale bar in the zoomed panels: 1 µm. f Quantification of CIP2A-TOPBP1 proximity by in situ PLA in U2OS cells transfected with either control siRNA (siCtrl) or TOPBP1 siRNA (siTOPBP1), arrested in mitosis by Nocodazole and mock treated or treated with 1 Gy of IR. Each data point represents one cell ( n = 61; pooled from two independent experiments), and bars represent median. Statistical significance was assessed by Kruskal-Wallis test and Dunn’s multiple comparison test (α = 0.05; ns = not significant). All scale bars = 10 µm unless indicated otherwise. Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: GFP (Mouse, Roche, 11814460001, 1:5000), GFP (Rabbit, Abcam, ab290, 1:1000), HA (Rabbit, Abcam, ab9110, 1:4000), MDC1 (Rabbit, Abcam, ab11171, 1:5000), MDC1 pSer168 (Rabbit, AMS Biotechnology, custom-made, 1:500) , MDC1 pSer196 (Rabbit, 21 st Century, custom-made 1:200) , TOPBP1 (Rabbit, Abcam, ab2402, 1:1500), CIP2A (Rabbit, Cell Signaling, 14805, 1:1000), CIP2A (Mouse, Santa Cruz, sc-80659, 1:1000), CRM1 (Rabbit, Atlas Antibodies, HPA042933, 1:1500), Tubulin (Mouse, Sigma, T6199, 1:5000), Tubulin hFABTM rhodamine (Bio-Rad, 12004166, 1:5000), FLAG M2 (Mouse, Sigma, F1804, 1:1000), pSer10 Histone H3 (Rabbit, Millipore, 06-570, 1:1000), Histone H3 (Rabbit, Abcam, ab1791, 1:10000).

Techniques: Staining, Irradiation, In Situ, Transfection, Control, Comparison

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

doi: 10.1038/s41467-022-31865-5

Figure Lengend Snippet: a Western blots of total cell extract of parental RPE-1 cells, RPE-1 ΔCIP2A cells and stably transduced RPE-1 ΔCIP2A cells with empty vector (+empty) and vector containing Flag-tagged wild type CIP2A cDNA (+Flag-WT). b Confocal micrographs (maximum intensity projections) of Nocodazole-arrested empty vector (+Empty) and Flag-tagged CIP2A wild type (+Flag-WT) complemented RPE-1 ΔCIP2A cells, treated with 1 Gy of IR and stained for TOPBP1 and CIP2A. c Quantification of the experiment in b . Number of CIP2A/TOPBP1 foci per cell was assessed (+Empty: n = 57, +Flag-WT: n = 62, pooled from three independent experiments). Statistical significance was assessed with the two-sided Mann-Whitney test (α = 0.05) d Western blots of total cell extracts of CIP2A knock-out RPE-1 cells stably transduced with Flag-tagged wild type CIP2A cDNA (RPE-1 ΔCIP2A + Flag-WT) and transiently transfected with control siRNA (siCtrl) and two different siRNAs against TOPBP1 (siTOPBP1 #1 and siTOPBP1 #2). e Confocal micrographs (maximum intensity projection) of CIP2A knock-out RPE-1 cells stably transduced with Flag-tagged wild type CIP2A cDNA (RPE-1 ΔCIP2A + Flag-WT) and transiently transfected with control siRNA (siCtrl) and two different siRNAs against TOPBP1 (siTOPBP1 #1 and siTOPBP1 #2). Cells were arrested in pro-metaphase with Nocodazole, fixed 1 h after 1 Gy of IR and stained for CIP2A and TOPBP1. f Quantification of the experiment in e . Number of CIP2A foci per cell was assessed (siCtrl: n = 62, siTOPBP1 #1: n = 46, siTOPBP1 #2: n = 44, pooled from three independent experiments). Bars and error bars represent mean and SD. Statistical significance was assessed with the Kruskal-Wallis test and Dunn’s multiple comparison test (α = 0.05). All scale bars = 10 µm. Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: GFP (Mouse, Roche, 11814460001, 1:5000), GFP (Rabbit, Abcam, ab290, 1:1000), HA (Rabbit, Abcam, ab9110, 1:4000), MDC1 (Rabbit, Abcam, ab11171, 1:5000), MDC1 pSer168 (Rabbit, AMS Biotechnology, custom-made, 1:500) , MDC1 pSer196 (Rabbit, 21 st Century, custom-made 1:200) , TOPBP1 (Rabbit, Abcam, ab2402, 1:1500), CIP2A (Rabbit, Cell Signaling, 14805, 1:1000), CIP2A (Mouse, Santa Cruz, sc-80659, 1:1000), CRM1 (Rabbit, Atlas Antibodies, HPA042933, 1:1500), Tubulin (Mouse, Sigma, T6199, 1:5000), Tubulin hFABTM rhodamine (Bio-Rad, 12004166, 1:5000), FLAG M2 (Mouse, Sigma, F1804, 1:1000), pSer10 Histone H3 (Rabbit, Millipore, 06-570, 1:1000), Histone H3 (Rabbit, Abcam, ab1791, 1:10000).

Techniques: Western Blot, Stable Transfection, Plasmid Preparation, Staining, MANN-WHITNEY, Knock-Out, Transduction, Transfection, Control, Comparison

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

doi: 10.1038/s41467-022-31865-5

Figure Lengend Snippet: a GFP-immunoprecipitation from 293FT cells either Mock transfected (Mock) or co-transfected with a GFP-tagged full-length TOPBP1 wild type (WT), and various deletion mutants as indicated, and Flag-tagged CIP2A. Relative intensities of co-immunoprecipitated Flag-CIP2A bands are indicated. b Confocal micrographs (maximum intensity projections) of Nocodazole-arrested GFP-TOPBP1 (WT and deletion mutants) expressing U2OS cells, treated with 1 Gy of IR. Endogenous TOPBP1 was depleted by 3′-UTR targeting TOPBP1 siRNA. c Quantification of GFP-TOPBP1/CIP2A foci per cell. Each data point represents one mitotic cell (WT: n = 29, ΔC: n = 30, Δ751–899: n = 31, Δ774–798: n = 31, Δ813–892: n = 30, Δ774–789 + Δ813-892: n = 32, pooled from three independent experiments). Statistical significance was assessed with the Kruskal-Wallis test and Dunn’s multiple comparison test (α = 0.05). All scale bars = 10 µm. Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: GFP (Mouse, Roche, 11814460001, 1:5000), GFP (Rabbit, Abcam, ab290, 1:1000), HA (Rabbit, Abcam, ab9110, 1:4000), MDC1 (Rabbit, Abcam, ab11171, 1:5000), MDC1 pSer168 (Rabbit, AMS Biotechnology, custom-made, 1:500) , MDC1 pSer196 (Rabbit, 21 st Century, custom-made 1:200) , TOPBP1 (Rabbit, Abcam, ab2402, 1:1500), CIP2A (Rabbit, Cell Signaling, 14805, 1:1000), CIP2A (Mouse, Santa Cruz, sc-80659, 1:1000), CRM1 (Rabbit, Atlas Antibodies, HPA042933, 1:1500), Tubulin (Mouse, Sigma, T6199, 1:5000), Tubulin hFABTM rhodamine (Bio-Rad, 12004166, 1:5000), FLAG M2 (Mouse, Sigma, F1804, 1:1000), pSer10 Histone H3 (Rabbit, Millipore, 06-570, 1:1000), Histone H3 (Rabbit, Abcam, ab1791, 1:10000).

Techniques: Immunoprecipitation, Transfection, Expressing, Comparison

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

doi: 10.1038/s41467-022-31865-5

Figure Lengend Snippet: a Confocal micrographs (maximum intensity projections) of Nocodazole-arrested RPE-1 wild type, MDC1 knock-out (ΔMDC1) and CIP2A knock-out (ΔCIP2A) cells, irradiated with 1 Gy and stained for TOPBP1 and CIP2A. b Quantification of the experiment in a . CIP2A/TOPBP1 foci were manually counted. Each data point represents one mitotic cell (no IR WT: n = 52, ΔMDC1: n = 56, ΔCIP2A: n = 51, IR WT: n = 80, ΔMDC1: n = 61, ΔCIP2A: n = 53, pooled from three independent experiments) and bars represent the median. Statistical significance was assessed with the Kruskal-Wallis test and Dunn’s multiple comparison test (α = 0.05). c Flag-immunoprecipitation from 293FT cells transfected with Flag-tagged full-length CIP2A and HA-tagged MDC1 wild type (WT) and Ser168/Ser196 double mutant (S168A/S196A). d Quantification of CIP2A-MDC1 proximity by in situ PLA in U2OS wild type (WT) and U2OS MDC1 knock-out cells (ΔMDC1), arrested in mitosis by Nocodazole and mock treated or treated with 1 Gy of IR. Each data point represents one cell (WT -IR: n = 131, WT + IR: n = 114, ΔMDC1 -IR: n = 85, ΔMDC1 + IR n = 54, pooled from two independent experiments), and bars represent median. Statistical significance was assessed by Kruskal-Wallis test and Dunn’s multiple comparison test (α = 0.05). e Confocal micrographs (maximum intensity projections) of Nocodazole-arrested U2OS ΔMDC1 cells stably transfected with GFP-tagged wild type and S168A/S196A mutated MDC1, stained for CIP2A and TOPBP1 1 h treatment with 1 Gy of IR. f Quantitative analysis of GFP-MDC1 and CIP2A co-localization by SQUASSH: Left graph: object size colocalization (area of object overlap divided by total object area). Right graph: object number colocalization (fraction of objects in each channel that overlap ≥ 50%). Data points represent individual mitotic cells (n = 24, except S168A/196 A object number: n = 21, pooled from three independent experiments). Bars and error bars represent mean and SD. Statistical significance was assessed by unpaired t-tests with Welch’s correction (α = 0.05). Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: GFP (Mouse, Roche, 11814460001, 1:5000), GFP (Rabbit, Abcam, ab290, 1:1000), HA (Rabbit, Abcam, ab9110, 1:4000), MDC1 (Rabbit, Abcam, ab11171, 1:5000), MDC1 pSer168 (Rabbit, AMS Biotechnology, custom-made, 1:500) , MDC1 pSer196 (Rabbit, 21 st Century, custom-made 1:200) , TOPBP1 (Rabbit, Abcam, ab2402, 1:1500), CIP2A (Rabbit, Cell Signaling, 14805, 1:1000), CIP2A (Mouse, Santa Cruz, sc-80659, 1:1000), CRM1 (Rabbit, Atlas Antibodies, HPA042933, 1:1500), Tubulin (Mouse, Sigma, T6199, 1:5000), Tubulin hFABTM rhodamine (Bio-Rad, 12004166, 1:5000), FLAG M2 (Mouse, Sigma, F1804, 1:1000), pSer10 Histone H3 (Rabbit, Millipore, 06-570, 1:1000), Histone H3 (Rabbit, Abcam, ab1791, 1:10000).

Techniques: Knock-Out, Irradiation, Staining, Comparison, Immunoprecipitation, Transfection, Mutagenesis, In Situ, Stable Transfection

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

doi: 10.1038/s41467-022-31865-5

Figure Lengend Snippet: a Western blots of total cell extract of RPE-1 wild type cells (WT) and RPE-1 CIP2A knock-out cells cells (ΔCIP2A) either Mock treated (-) or treated with the PP2A inhibitor LB-100. b GFP pull-down from 293FT cells transfected with GFP-tagged full-length MDC1 and either control siRNA (siCtrl) or siRNA against CIP2A (siCIP2A). All scale bars = 10 µm. Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: GFP (Mouse, Roche, 11814460001, 1:5000), GFP (Rabbit, Abcam, ab290, 1:1000), HA (Rabbit, Abcam, ab9110, 1:4000), MDC1 (Rabbit, Abcam, ab11171, 1:5000), MDC1 pSer168 (Rabbit, AMS Biotechnology, custom-made, 1:500) , MDC1 pSer196 (Rabbit, 21 st Century, custom-made 1:200) , TOPBP1 (Rabbit, Abcam, ab2402, 1:1500), CIP2A (Rabbit, Cell Signaling, 14805, 1:1000), CIP2A (Mouse, Santa Cruz, sc-80659, 1:1000), CRM1 (Rabbit, Atlas Antibodies, HPA042933, 1:1500), Tubulin (Mouse, Sigma, T6199, 1:5000), Tubulin hFABTM rhodamine (Bio-Rad, 12004166, 1:5000), FLAG M2 (Mouse, Sigma, F1804, 1:1000), pSer10 Histone H3 (Rabbit, Millipore, 06-570, 1:1000), Histone H3 (Rabbit, Abcam, ab1791, 1:10000).

Techniques: Western Blot, Knock-Out, Transfection, Control

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

doi: 10.1038/s41467-022-31865-5

Figure Lengend Snippet: a Confocal micrographs (maximum intensity projections) of Leptomycin B, Selinexor and control DMSO treated U2OS cells, stained for CIP2A. b Quantification of nuclear CIP2A staining of the experiment in a . Each data point corresponds to one cell (DMSO: n = 173, LeptomycinB: n = 150, Selinexor: n = 178, pooled from two independent experiments). Bars and error bars represent mean and SD. Statistical significance was calculated using one-way ANOVA and Dunnett’s multiple comparison test (α = 0.05). c Confocal micrographs (maximum intensity projections) of RPE-1 ΔCIP2A cells and RPE-1 ΔCIP2A cells stably transduced with Flag-tagged full-length CIP2A (WT) and CIP2A deletion mutant (human CIP2A amino acids 561–625; ΔNES), stained for CIP2A. d Quantification of nuclear CIP2A staining of the experiment in c . Each data point corresponds to one cell (+Epmpty: n = 199, +Flag-WT: n = 155, +Flag-ΔNES: n = 165, pooled from two independent experiments). Bars and error bars represent mean and SD. Statistical significance between +Flag-WT and +Flag-ΔNES was calculated using two-sided Welch’s t -test (α = 0.05). e Flag-immunoprecipitation from 293FT cells either Mock transfected (Mock) or transfected with a Flag-tagged full-length CIP2A wild type (WT), and Flag-tagged CIP2A lacking amino acids 561-625 (ΔNES). Relative intensities of co-immunoprecipitated CRM1 bands are indicated. f Confocal micrograph (maximum intensity projection) of Hela cells expressing endogenous Clover-tagged LMNA and stained for CIP2A. g Quantification of CIP2A-TOPBP1 proximity by in situ PLA in U2OS cells transfected with control siRNA (siCtrl) and siRNA against TOPBP1 (siTOPBP1), arrested in mitosis by Nocodazole and treated with 1 Gy of IR. Interphase cells (I) were separated from mitotic cells (M) with an automatic image analysis pipeline, based on DAPI mean intensities. Each data point represents one cell (siCtrl I: n = 92, siCtrl M: n = 63, siTOPBP1 I: n = 138, siTOPBP1 M: n = 121; pooled from two independent experiments), and bars represent median. Statistical significance was assessed by Kruskal-Wallis test and Dunn’s multiple comparison test (α = 0.05). Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: GFP (Mouse, Roche, 11814460001, 1:5000), GFP (Rabbit, Abcam, ab290, 1:1000), HA (Rabbit, Abcam, ab9110, 1:4000), MDC1 (Rabbit, Abcam, ab11171, 1:5000), MDC1 pSer168 (Rabbit, AMS Biotechnology, custom-made, 1:500) , MDC1 pSer196 (Rabbit, 21 st Century, custom-made 1:200) , TOPBP1 (Rabbit, Abcam, ab2402, 1:1500), CIP2A (Rabbit, Cell Signaling, 14805, 1:1000), CIP2A (Mouse, Santa Cruz, sc-80659, 1:1000), CRM1 (Rabbit, Atlas Antibodies, HPA042933, 1:1500), Tubulin (Mouse, Sigma, T6199, 1:5000), Tubulin hFABTM rhodamine (Bio-Rad, 12004166, 1:5000), FLAG M2 (Mouse, Sigma, F1804, 1:1000), pSer10 Histone H3 (Rabbit, Millipore, 06-570, 1:1000), Histone H3 (Rabbit, Abcam, ab1791, 1:10000).

Techniques: Control, Staining, Comparison, Stable Transfection, Transduction, Mutagenesis, Immunoprecipitation, Transfection, Expressing, In Situ

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

doi: 10.1038/s41467-022-31865-5

Figure Lengend Snippet: a Clonogenic survival analysis of IR treated RPE-1 wild type (WT) cells and RPE-1 CIP2A knock-out cells (ΔCIP2A). Data points represent the mean of 3 independent experiments, error bars represent the SD. Statistical significance was assessed by linear regression (α = 0.05). b Quantification of MNi formation, c quantification of CENPA positive (CENPA + ) and CENPA negative (CENPA-) as well as γH2AX positive (γH2AX + ) and γH2AX negative (γH2AX-) MNi, d quantification of residual γH2AX foci 24 h after irradiation of mitotic cells and e quantification of chromosomal aberrations in metaphase spreads in RPE-1 parental cells (RPE-1), RPE-1 MDC1 knock-out cells (RPE-1 ΔMDC1), RPE-1 CIP2A knock-out cells (RPE-1 ΔCIP2A) and RPE-1 CIP2A knock-out cells stably transduced with empty vector (+ empty) and Flag-tagged wild type CIP2A ( + Flag-WT). Columns in b represent mean of 3–4 independent experiments (RPE-1 ± IR, RPE-1 ΔMDC1 ± IR, + empty -IR, + Flag-WT -IR: n = 3, +empty +IR, + Flag-WT + IR: n = 4), data points represent percentage of cells with MNi in the experiments. Parts of whole are displayed for each cell line in c . The numbers of MNi assessed for each cell line (n) in c are indicated above each column. Each data point in d represents log of total γH2AX foci intensity per cell (RPE-1: n = 368; RPE-1 ΔMDC1: n = 330; +Empty: n = 219; +Flag-WT: n = 224), bars and error bars represent mean and SD. Statistical significance was assessed by one-way ANOVA and Sidak’s multiple comparison test (α = 0.05) ns: not significant. Aberrations in e were counted manually. Each data point represents one metaphase (RPE-1: n = 16, RPE-1 ΔMDC1: n = 12, RPE-1 ΔCIP2A: n = 19, +Empty: n = 13, +Flag-WT: n = 13), bars represent the median. Statistical significance was assessed by the Kruskal-Wallis test and Dunn’s multiple comparison test (α = 0.05). ns: not significant. f Examples of chromosomal aberrations in metaphase spreads derived from RPE-1 parental (RPE-1) and RPE-1 CIP2A knock-out cells (ΔCIP2A). Aberrations including single chromatid telomere loss, sister chromatid telomere loss, interstitial telomeres, telomere duplications and dicentric chromosomes are indicated with white arrowheads. For d , e : One representative of two independent experiments is shown. Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: GFP (Mouse, Roche, 11814460001, 1:5000), GFP (Rabbit, Abcam, ab290, 1:1000), HA (Rabbit, Abcam, ab9110, 1:4000), MDC1 (Rabbit, Abcam, ab11171, 1:5000), MDC1 pSer168 (Rabbit, AMS Biotechnology, custom-made, 1:500) , MDC1 pSer196 (Rabbit, 21 st Century, custom-made 1:200) , TOPBP1 (Rabbit, Abcam, ab2402, 1:1500), CIP2A (Rabbit, Cell Signaling, 14805, 1:1000), CIP2A (Mouse, Santa Cruz, sc-80659, 1:1000), CRM1 (Rabbit, Atlas Antibodies, HPA042933, 1:1500), Tubulin (Mouse, Sigma, T6199, 1:5000), Tubulin hFABTM rhodamine (Bio-Rad, 12004166, 1:5000), FLAG M2 (Mouse, Sigma, F1804, 1:1000), pSer10 Histone H3 (Rabbit, Millipore, 06-570, 1:1000), Histone H3 (Rabbit, Abcam, ab1791, 1:10000).

Techniques: Knock-Out, Irradiation, Stable Transfection, Transduction, Plasmid Preparation, Comparison, Derivative Assay

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: Immunohistochemical staining was conducted with anti-CIP2A rabbit polyclonal antibody (Novus Biologicals; 1:100), anti-Plk1 rabbit polyclonal antibody (Abcam; 1:100), or anti-phospho-Plk1 rabbit monoclonal antibody (Abcam; 1:100).

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: Immunohistochemical staining was conducted with anti-CIP2A rabbit polyclonal antibody (Novus Biologicals; 1:100), anti-Plk1 rabbit polyclonal antibody (Abcam; 1:100), or anti-phospho-Plk1 rabbit monoclonal antibody (Abcam; 1:100).

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: Immunohistochemical staining was conducted with anti-CIP2A rabbit polyclonal antibody (Novus Biologicals; 1:100), anti-Plk1 rabbit polyclonal antibody (Abcam; 1:100), or anti-phospho-Plk1 rabbit monoclonal antibody (Abcam; 1:100).

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: Immunohistochemical staining was conducted with anti-CIP2A rabbit polyclonal antibody (Novus Biologicals; 1:100), anti-Plk1 rabbit polyclonal antibody (Abcam; 1:100), or anti-phospho-Plk1 rabbit monoclonal antibody (Abcam; 1:100).

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: Immunohistochemical staining was conducted with anti-CIP2A rabbit polyclonal antibody (Novus Biologicals; 1:100), anti-Plk1 rabbit polyclonal antibody (Abcam; 1:100), or anti-phospho-Plk1 rabbit monoclonal antibody (Abcam; 1:100).

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

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 6. Association between CIP2A and Plk1 in lung, gastric, colon, and cervical cancers and their normal tissue counterparts. A, quantification of CIP2A and Plk1 staining intensities in lung, gastric, colon, and cervical cancers and their normal tissue counterparts. B and C, representative microscopic images of lung, gastric, colon, and cervical cancers and their normal tissue counterparts stained with anti-CIP2A antibody (B) or anti-Plk1 antibody (C). Scale bars, 200 mm. D, representative high-magnification images of gastric cancer tissues stained with the indicated antibodies. Scale bars, 100 mm. E and F, tissue sections were incubated with rabbit anti-CIP2A antibody together with mouse anti-Plk1 antibody followed by in situ PLA analysis. Representative confocal images of lung, gastric, colon, and cervical cancers and their normal tissue counterparts (E, left and F). Scale bars, 20 mm. Four different areas were obtained for each sample and 200 to 500 cells were quantified per area (mm2) using CellProfiler (E, right). Nuclei were stained with Hoechst (blue). The green signal represents autofluorescence. N, normal lung tissue. C, cancer tissue. A and E (left), data are presented as box-and-whisker plots. , P < 0.001 compared with their normal tissue counterparts.

Article Snippet: Immunohistochemical staining was conducted with anti-CIP2A rabbit polyclonal antibody (Novus Biologicals; 1:100), anti-Plk1 rabbit polyclonal antibody (Abcam; 1:100), or anti-phospho-Plk1 rabbit monoclonal antibody (Abcam; 1:100).

Techniques: Staining, Incubation, In Situ, Whisker Assay

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic RAS promotes MYC protein stability by upregulating the expression of the inhibitor of apoptosis protein family member Survivin

doi: 10.1016/j.jbc.2022.102842

Figure Lengend Snippet: Survivin maintains the phosphorylation of Ser 62 in MYC. A , representative Western blot analysis of Survivin, MYC, phospho-MYC (P-MYC) Ser 62, and P-MYC Thr 58 levels in PANC-1 and MIA PaCa-2 cells treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155 for either 8 or 24 h. The relative changes in MYC phosphorylation at Ser 62 and Thr 58 were quantified using densitometry and listed below the corresponding blots. B , quantification of the relative changes in the phosphorylation of MYC at Ser 62 in cells treated without (DMSO) or with YM155 for 8 h. C , representative Western blot analysis of PP2A-A, PP2A-B, and PP2A-C expression in PANC-1 and MIA PaCa-2 cells treated without (DMSO) or with 250 nM YM155. D , representative Western blot analysis of Survivin and CIP2A expression in PANC-1 and MIA PaCa-2 cells treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155. E , representative Western blot analysis of CIP2A and MYC expression in PANC-1 cells expressing negative control (NC) and CIP2A-targeting shRNAs. F , representative Western blot analysis of Survivin and V5-tagged CIP2A expression in PANC-1 cells ectopically expressing V5-tagged CIP2A and treated with the indicated combinations of 25 μM chloroquine (CQ) and 250 nM YM155. G , quantitative PCR (qPCR) analysis of CIP2A mRNA levels, relative to actin mRNA levels, in PANC-1 and MIA PaCa-2 cells treated without (DMSO) or with 250 nM YM155 for 24 h. In ( A, C – F ), vinculin was used as the loading controls. All experiments involving Western blots were performed a minimum of three independent times, with similar results being obtained each time. In ( B and F ), data are means ± standard errors of at least three experiments; ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by Student’s t test. PP2A, protein phosphatase 2A.

Article Snippet: The human PANC-1 and MIA PaCa-2 PDAC cell lines, as well as the MEFs, were grown in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum. pCDNA3 expression constructs encoding Myc-tagged Survivin (Sino Biological), HA-tagged MYC (Addgene #74164) , V5-tagged CIP2A (Addgene #119287) , GFP-tagged MYC (Addgene #42142) , and mCherry-LC3B (Addgene #40827) ( ) were transfected into MEFs using PEI (Sigma) and PDAC cells using FuGENE 6 (Promega) per the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Negative Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic RAS promotes MYC protein stability by upregulating the expression of the inhibitor of apoptosis protein family member Survivin

doi: 10.1016/j.jbc.2022.102842

Figure Lengend Snippet: Diagram showing how KRAS-mediated upregulation of Survivin expression stabilizes MYC to promote PDAC cell growth. In normal cells, WT KRAS transiently activates ERK1/2, which in turn leads to the phosphorylation of Ser 62 (S62) in MYC, maintaining its stability. MYC is subsequently phosphorylated on Thr 58 (T58) by GSK-3β and dephosphorylated on Ser 62 by PP2A, which targets MYC for degradation (left side of diagram). However, mutant forms of KRAS (KRAS mt ) potently activate ERK1/2 and increase the phosphorylation of Ser 62 in MYC, enhancing its stability. We also found that mutant KRAS-induced ERK activation strongly upregulates Survivin expression, which further enhances MYC expression by both inhibiting its autophagy-dependent degradation, as well as by promoting the expression of CIP2A, preventing PP2A from dephosphorylating Ser 62 in MYC. These effects ensure that the proper amount of MYC is expressed in PDAC cells to support their optimal growth and to work together with KRAS to drive oncogenic transformation (right side of diagram). This figure was created using BioRender.com . ERK1/2, extracellular signal–regulated kinase 1/2; GSK-3β, glycogen synthase kinase 3β; PP2A, protein phosphatase 2A.

Article Snippet: The human PANC-1 and MIA PaCa-2 PDAC cell lines, as well as the MEFs, were grown in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum. pCDNA3 expression constructs encoding Myc-tagged Survivin (Sino Biological), HA-tagged MYC (Addgene #74164) , V5-tagged CIP2A (Addgene #119287) , GFP-tagged MYC (Addgene #42142) , and mCherry-LC3B (Addgene #40827) ( ) were transfected into MEFs using PEI (Sigma) and PDAC cells using FuGENE 6 (Promega) per the manufacturer’s instructions.

Techniques: Expressing, Mutagenesis, Activation Assay, Transformation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: CIP2A facilitates the G1/S cell cycle transition via B‐Myb in human papillomavirus 16 oncoprotein E6‐expressing cells

doi: 10.1111/jcmm.13693

Figure Lengend Snippet: Induction of CIP2A mRNA and protein expression by HPV‐16E6 in PHKs. A, mRNA expression of HPV‐16E6 in PHKs expressing 16E6 and F2V using β‐actin as a loading control. B, Protein levels of HPV‐16E6, p53 and p21 in PHKs expressing 16E6 and F2V. Expression of GAPDH was used as a loading control. A representative of 2 independent experiments is shown. C, HPV‐16E6 expression leads to increased protein expression of CIP2A in PHKs. Data from a representative of 3 experiments are shown. D, Data from 3 experiments are summarized. E, Relative CIP2A mRNA expression was determined by qRT‐PCR in the above cells. Data from 3 experiments are summarized. The mean and standard deviation (SD) of 3 independent experiments are shown. Babe, pBabe‐puromycin vector. *, P < .05; **, P < .01; and ***, P < .001

Article Snippet: Membranes were probed with primary antibodies specific for the following proteins: Cdk1 (BD Biosciences, 610038), p21 (BD Biosciences, 610233); CIP2A (Novus Biologicals, NB100‐68264); B‐Myb (sc‐725), Cdk2 (sc‐6248), Cdk4 (sc‐260), Cdk6 (sc‐177), cyclin A2 (sc‐751), cyclin B1 (sc‐752), cyclin D1 (sc‐718), cyclin E1 (sc‐198), c‐Myc (sc‐764), p53 (sc‐126) and GAPDH (all from Santa Cruz Biotechnology, sc‐25778); and phospho‐c‐Myc (phospho S62, Abcam, ab51156), HPV‐16/18E6 (Abcam 70) and β‐tubulin (Sigma‐Aldrich, T4026).

Techniques: Expressing, Control, Quantitative RT-PCR, Standard Deviation, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: CIP2A facilitates the G1/S cell cycle transition via B‐Myb in human papillomavirus 16 oncoprotein E6‐expressing cells

doi: 10.1111/jcmm.13693

Figure Lengend Snippet: Inhibition of CIP2A by siRNA impeded cell viability and DNA synthesis in HPV‐16E6–expressing cells. A, Elevated expression of CIP2A protein in 16E6‐expressing RPE1 cells. B, Western blot analysis of CIP2A and p53 proteins after transfection with scrambled siRNA (siCon) or CIP2A siRNA (siCIP2A) for 48 h. A representative of 3 independent experiments is shown. C, Cell viability assay of RPE1‐16E6 cells with CIP2A knockdown. D, Representative flow cytometry of BrdU staining profiles is shown. E, The mean and SD of BrdU‐positive cells from 3 experiments are summarized. **, P < .01.

Article Snippet: Membranes were probed with primary antibodies specific for the following proteins: Cdk1 (BD Biosciences, 610038), p21 (BD Biosciences, 610233); CIP2A (Novus Biologicals, NB100‐68264); B‐Myb (sc‐725), Cdk2 (sc‐6248), Cdk4 (sc‐260), Cdk6 (sc‐177), cyclin A2 (sc‐751), cyclin B1 (sc‐752), cyclin D1 (sc‐718), cyclin E1 (sc‐198), c‐Myc (sc‐764), p53 (sc‐126) and GAPDH (all from Santa Cruz Biotechnology, sc‐25778); and phospho‐c‐Myc (phospho S62, Abcam, ab51156), HPV‐16/18E6 (Abcam 70) and β‐tubulin (Sigma‐Aldrich, T4026).

Techniques: Inhibition, DNA Synthesis, Expressing, Western Blot, Transfection, Viability Assay, Knockdown, Flow Cytometry, BrdU Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: CIP2A facilitates the G1/S cell cycle transition via B‐Myb in human papillomavirus 16 oncoprotein E6‐expressing cells

doi: 10.1111/jcmm.13693

Figure Lengend Snippet: Silencing CIP2A caused G1 arrest in 16E6‐expressing cells. A, Flow cytometric analysis of 16E6‐expressing cells transfected with CIP2A siRNA for 36 h, treated with PBS or 10 μg/mL bleomycin for 24 h and then stained with PI. G1, S and G2 phases are indicated. Data from a representative of 4 experiments are shown. B, Quantification of percentages G1 phase and S phase cells. Data from 4 experiments are summarized. *, P < .05; **, P < .01

Article Snippet: Membranes were probed with primary antibodies specific for the following proteins: Cdk1 (BD Biosciences, 610038), p21 (BD Biosciences, 610233); CIP2A (Novus Biologicals, NB100‐68264); B‐Myb (sc‐725), Cdk2 (sc‐6248), Cdk4 (sc‐260), Cdk6 (sc‐177), cyclin A2 (sc‐751), cyclin B1 (sc‐752), cyclin D1 (sc‐718), cyclin E1 (sc‐198), c‐Myc (sc‐764), p53 (sc‐126) and GAPDH (all from Santa Cruz Biotechnology, sc‐25778); and phospho‐c‐Myc (phospho S62, Abcam, ab51156), HPV‐16/18E6 (Abcam 70) and β‐tubulin (Sigma‐Aldrich, T4026).

Techniques: Expressing, Transfection, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: CIP2A facilitates the G1/S cell cycle transition via B‐Myb in human papillomavirus 16 oncoprotein E6‐expressing cells

doi: 10.1111/jcmm.13693

Figure Lengend Snippet: Silencing CIP2A caused decreased Cdk1 and Cdk2 proteins in 16E6‐expressing cells. A, Western blot analysis of CIP2A, Cdk4, Cdk6, cyclin D1, Cdk1, Cdk2, cyclin B1, cyclin A2 and cyclin E1 protein levels in cells expressing HPV‐16E6 transfected with CIP2A siRNA and then treated with PBS or 10 μg/mL bleomycin for 24 h. A representative of 3 independent experiments is shown. B, Quantification of all cell cycle‐related proteins. Data from 3 experiments are summarized. C, Relative mRNA levels of all cell cycle‐related genes determined by qRT‐PCR. Data from 3 experiments are summarized. *, P < .05; **, P < .01

Article Snippet: Membranes were probed with primary antibodies specific for the following proteins: Cdk1 (BD Biosciences, 610038), p21 (BD Biosciences, 610233); CIP2A (Novus Biologicals, NB100‐68264); B‐Myb (sc‐725), Cdk2 (sc‐6248), Cdk4 (sc‐260), Cdk6 (sc‐177), cyclin A2 (sc‐751), cyclin B1 (sc‐752), cyclin D1 (sc‐718), cyclin E1 (sc‐198), c‐Myc (sc‐764), p53 (sc‐126) and GAPDH (all from Santa Cruz Biotechnology, sc‐25778); and phospho‐c‐Myc (phospho S62, Abcam, ab51156), HPV‐16/18E6 (Abcam 70) and β‐tubulin (Sigma‐Aldrich, T4026).

Techniques: Expressing, Western Blot, Transfection, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: CIP2A facilitates the G1/S cell cycle transition via B‐Myb in human papillomavirus 16 oncoprotein E6‐expressing cells

doi: 10.1111/jcmm.13693

Figure Lengend Snippet: Regulation of Cdk1 and Cdk2 by CIP2A is dependent on B‐Myb rather than c‐Myc. A, Western blot analysis of CIP2A, c‐Myc, phospho‐S62‐Myc and B‐Myb protein levels in 16E6‐expressing cells after CIP2A knockdown. β‐Tubulin was used as a loading control. B, Protein levels of B‐Myb, c‐Myc and phospho‐S62‐Myc in 16E6‐expressing PHKs and (C) RPE1 cells. D, Protein levels of B‐Myb, Cdk1 and Cdk2, CIP2A and p53 in 16E6‐expressing cells after B‐Myb knockdown with siRNA. Data from a representative of 3 experiments are shown. E, Knockdown of B‐Myb down‐regulates Cdk1 and Cdk2 luciferase reporter activities. RPE1 cells were cotransfected with the Cdk1 or Cdk2 promoter‐luciferase constructs and renilla luciferase control plasmid together with B‐Myb siRNA plasmid. Cells were harvested after 48 h, and lysates were assayed for luciferase activity. F, Flow cytometric analysis of 16E6‐expressing cells transfected with B‐Myb siRNA treated with PBS or bleomycin. G1, S and G2 phases are indicated. A representative flow cytometry of 3 independent experiments is shown. G, Quantification of percentages G1 phase cells. Data from 3 experiments are summarized. H, Western blot analysis of B‐Myb, Cdk1 and Cdk2 in B‐Myb–overexpressing CIP2A knockdown cells. Data from a representative of 3 experiments are shown. *, P < .05; ***, P < .001

Article Snippet: Membranes were probed with primary antibodies specific for the following proteins: Cdk1 (BD Biosciences, 610038), p21 (BD Biosciences, 610233); CIP2A (Novus Biologicals, NB100‐68264); B‐Myb (sc‐725), Cdk2 (sc‐6248), Cdk4 (sc‐260), Cdk6 (sc‐177), cyclin A2 (sc‐751), cyclin B1 (sc‐752), cyclin D1 (sc‐718), cyclin E1 (sc‐198), c‐Myc (sc‐764), p53 (sc‐126) and GAPDH (all from Santa Cruz Biotechnology, sc‐25778); and phospho‐c‐Myc (phospho S62, Abcam, ab51156), HPV‐16/18E6 (Abcam 70) and β‐tubulin (Sigma‐Aldrich, T4026).

Techniques: Western Blot, Expressing, Knockdown, Control, Luciferase, Construct, Plasmid Preparation, Activity Assay, Transfection, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: CIP2A facilitates the G1/S cell cycle transition via B‐Myb in human papillomavirus 16 oncoprotein E6‐expressing cells

doi: 10.1111/jcmm.13693

Figure Lengend Snippet: Inhibition of Cdk1 and Cdk2 by CIP2A knockdown in cervical cancer SiHa cells caused G1 arrest. A, Western blot analysis of 16E6, p53 and CIP2A after HPV‐16E6 knockdown in cervical cancer SiHa cells. B, Protein expression of CIP2A, B‐Myb, Cdk1 and Cdk2 in SiHa cells after CIP2A knockdown. Data from a representative of 3 experiments are shown. C, Flow cytometric analysis of SiHa cells with CIP2A knockdown treated with PBS or bleomycin. A representative flow cytometry of 3 independent experiments is shown. D, Quantification of percentages G1 phase cells. Data from 3 experiments are summarized. *, P < .05

Article Snippet: Membranes were probed with primary antibodies specific for the following proteins: Cdk1 (BD Biosciences, 610038), p21 (BD Biosciences, 610233); CIP2A (Novus Biologicals, NB100‐68264); B‐Myb (sc‐725), Cdk2 (sc‐6248), Cdk4 (sc‐260), Cdk6 (sc‐177), cyclin A2 (sc‐751), cyclin B1 (sc‐752), cyclin D1 (sc‐718), cyclin E1 (sc‐198), c‐Myc (sc‐764), p53 (sc‐126) and GAPDH (all from Santa Cruz Biotechnology, sc‐25778); and phospho‐c‐Myc (phospho S62, Abcam, ab51156), HPV‐16/18E6 (Abcam 70) and β‐tubulin (Sigma‐Aldrich, T4026).

Techniques: Inhibition, Knockdown, Western Blot, Expressing, Flow Cytometry

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: A. Positive expression of CIP2A in pancreatic cancer tissue(X400). B. Negative expression of CIP2A in pancreatic cancer tissue(X400). C. Negative expression of CIP2A in adjacent normal pancreatic tissue(X400). D. Positive expression of CIP2A in adjacent normal pancreatic tissue(X400) E. Correlation of CIP2A expression and overall survival in all patients. F. Correlation of CIP2A expression and overall survival in stage II patients.

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Expressing

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: Expression of CIP2A in PDAC and adjacent normal pancreatic tissue [n, n(%)]

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Expressing

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: CIP2A protein expression and clinicopathologic features in PDAC patients

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Expressing

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: A. Relative CIP2A mRNA level in pancreatic cancer cell lines. B. Expression of CIP2A protein in pancreatic cancer cell lines. C. The mRNA levels of CIP2A in SW1990 cells after CIP2A-sh transfection. D. The mRNA levels of CIP2A in SW1990/R cells after CIP2A-sh transfection E. Western blot detection of CIP2A protein in pancreatic cancer cell lines after CIP2A-sh transfection. (* = p ≤ 0.05).

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Expressing, Transfection, Western Blot

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: IC50 decreased after CIP2A knock-down in both SW1990 A. and SW1990/R B. cells. C. Flow cytometric analysis using PI and Annexin V staining after gemcitabine treatments at different concentrations in SW1990 and SW1990-CIP2A cells. D. Flow cytometric analysis after gemcitabine treatments at different concentrations in SW1990/R and SW1990/R-CIP2A cells (* = p ≤ 0.05).

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Staining

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: Knockdown of CIP2A decreased AKT and BCL2 at mRNA level in both SW1990 A. and SW1990/R B. cells. Western blot C. showed decreased protein level of p-AKT and BLC2. (* = p ≤ 0.05).

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Western Blot

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: A. Positive expression of CIP2A in pancreatic cancer tissue(X400). B. Negative expression of CIP2A in pancreatic cancer tissue(X400). C. Negative expression of CIP2A in adjacent normal pancreatic tissue(X400). D. Positive expression of CIP2A in adjacent normal pancreatic tissue(X400) E. Correlation of CIP2A expression and overall survival in all patients. F. Correlation of CIP2A expression and overall survival in stage II patients.

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Expressing

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: Expression of CIP2A in PDAC and adjacent normal pancreatic tissue [n, n(%)]

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Expressing

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: CIP2A protein expression and clinicopathologic features in PDAC patients

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Expressing

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: A. Relative CIP2A mRNA level in pancreatic cancer cell lines. B. Expression of CIP2A protein in pancreatic cancer cell lines. C. The mRNA levels of CIP2A in SW1990 cells after CIP2A-sh transfection. D. The mRNA levels of CIP2A in SW1990/R cells after CIP2A-sh transfection E. Western blot detection of CIP2A protein in pancreatic cancer cell lines after CIP2A-sh transfection. (* = p ≤ 0.05).

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Expressing, Transfection, Western Blot

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: IC50 decreased after CIP2A knock-down in both SW1990 A. and SW1990/R B. cells. C. Flow cytometric analysis using PI and Annexin V staining after gemcitabine treatments at different concentrations in SW1990 and SW1990-CIP2A cells. D. Flow cytometric analysis after gemcitabine treatments at different concentrations in SW1990/R and SW1990/R-CIP2A cells (* = p ≤ 0.05).

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Knockdown, Staining

Journal: Oncotarget

Article Title: CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

doi: 10.18632/oncotarget.7447

Figure Lengend Snippet: Knockdown of CIP2A decreased AKT and BCL2 at mRNA level in both SW1990 A. and SW1990/R B. cells. Western blot C. showed decreased protein level of p-AKT and BLC2. (* = p ≤ 0.05).

Article Snippet: Three human CIP2A pshRNA plasmids (sh1, sh2, and sh3) were designed against three different CIP2A targets and constructed in pshRNA-iTS1-puro vector (Genloci) for SW1990 and in pshRNA-iTS1-G418 vector (Genloci) for SW1990/R.

Techniques: Knockdown, Western Blot