Journal: Autophagy

Article Title: Long-lived mice with reduced growth hormone signaling have a constitutive upregulation of hepatic chaperone-mediated autophagy

doi: 10.1080/15548627.2020.1725378

Figure Lengend Snippet: Snell dwarf mice have a reduction in the levels of CMA substrate CIP2A in the liver and kidney. (A) Representative western blots of whole liver lysate, showing relative protein levels of CIP2A, MYC, and the three PPP2 subunits (the antibody for subunit A recognizes PPP2R1A and PPP2R1B; the antibody for subunit B recognizes PPP2R2A, PPP2R2B, PPP2R2G, PPP2R2D, and the antibody subunit C recognizes PPP2CA and PPP2CB). (B) Western blot of isolated lysosomes from the liver of fed Snell and sibling control mice and ghr KO and sibling control mice injected with leupeptin or PBS control, showing accumulation of CIP2A in lysosomes of Snell and ghr KO mice. (C) Quantification of relative protein levels of CIP2A, MYC, and the PPP2 subunits from the liver, as shown in (A), normalized to the sibling controls. (D) Quantification of relative protein levels of CIP2A, MYC, and the PPP2 subunits from kidney, normalized to the sibling controls. (E & F) Relative mRNA levels of Myc and Cip2a mRNA from liver and kidney, respectively. Gapdh is shown as a control. Error bars are S.E.M. Where applicable, significant t-test results are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The following antibodies were used: ACTB (Cell Signaling Technologies; CST, 4967L), PPP2 subunit A (CST, 2039S; recognizes PPP2R1A and PPP2R1B), PPP2 subunit B (CST, 2290S; recognizes PPP2R2A, PPP2R2B, PPP2R2G, PPP2R2D), PPP2 subunit C (CST, 2259S; recognizes PPP2CA and PPP2CB), ENO1 (CST, 3810S), GAPDH (CST, 2118S), ACADL (AbCam, ab196655), MAP1LC3B (CST, 2775S), CTSD (AbCam, ab75852), LAMP1 (AbCam, ab24170), LAMP2A (AbCam, ab125068), CIP2A (Novus Biologicals, NB110-59722 & NB100-68264), MYC (AbCam, ab32072), GFAP (AbCam, ab7260), pS8 GFAP (ThermoFisher, PA5-12991), EEF1A (AbCam, ab37969), MAPT (CST, 46687S).

Techniques: Western Blot, Isolation, Control, Injection

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: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: (A) RSC96 cells were incubated with GAS6 (100 ng/ml), and immunoblotting evaluations of CIP2A and p-DAPK expression were performed. The lower panel shows the intensity during basal conditions from the scanned imaged (upper panel). (B) RSC96 cells incubated with and without GAS6 (100 ng/ml) for 30 min and analysed by immunofluorescence. (C) Immunohistochemistry images demonstrated that CIP2A expression levels were higher than sham levels at 14 days and 28 days in the cavernous nerve after BCNI.

Article Snippet: CIP2A cDNA (KIAA1524) was purchased from Origene (Rockville, MD).

Techniques: Incubation, Western Blot, Expressing, Immunofluorescence, Immunohistochemistry

Journal: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: (A-B) Co-immunoprecipitation between p-Axl, p-DAPK and CIP2A with GAS6 stimulation for 30 min in RSC96 cells. Total lysate indicates 1/10 input in each experiment. The relative quantification of protein expression was normalized with respect to β-actin expression. (C) Immunofluorescence image demonstrating co-localized CIP2A and p-Axl and co-localized p-Axl and p-DAPK (D) after GAS6 stimulation for 30 min in RSC96 cells.

Article Snippet: CIP2A cDNA (KIAA1524) was purchased from Origene (Rockville, MD).

Techniques: Immunoprecipitation, Quantitative Proteomics, Expressing, Immunofluorescence

Journal: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: (A) RSC96 cells were transfected with either control or DAPK siRNA for 48 h and then exposed to GAS6 (100 ng/ml) for 30 min. Immunoblotting evaluations of pAxl, Axl, CIP2A, DAPK, pERK1/2, ERK1/2, pAKT, AKT, Myc and Survivin. (B) RSC96 cells were transfected with either control or CIP2A siRNA for 48 h and then exposed to GAS6 (100 ng/ml) for 30 min. Immunoblotting evaluations of pAxl, Axl, DAPK, pDAPK, pERK1/2, ERK1/2, pAKT, AKT, Myc and Survivin. (C) DAPK or CIP2A was knocked down for 48 h then RSC96 cells were incubated with GAS6 (100 ng/ml) at the indicated hours. Cell viability was analysed via the WST-1 assay. Data are the mean ± SD, and n = 3 for each time point. * p < 0.05, ** p < 0.01 vs. scramble.

Article Snippet: CIP2A cDNA (KIAA1524) was purchased from Origene (Rockville, MD).

Techniques: Transfection, Control, Western Blot, Incubation, WST-1 Assay

Journal: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: RSC96 cells were transfected with control, CIP2A siRNA (A) , DKK-CIP2A (B) , or DAPK siRNA (C) for 48 h and then exposed to GAS6 (100 ng/ml) for 30 min. PP2A activity and immunoblotting evaluations of DKK, CIP2A, pDAPK, and DAPK were performed. (D) RSC96 cells were incubated with GAS6 (100 ng/ml) for 30 min and co-immunoprecipitation between CIP2A, pDAPK and PP2A was evaluated. Immunoprecipitated PP2A activity was measured. Data are the mean ± SD, and n = 3 for each time point. * p < 0.05, ** p < 0.01, vs. control.

Article Snippet: CIP2A cDNA (KIAA1524) was purchased from Origene (Rockville, MD).

Techniques: Transfection, Control, Activity Assay, Western Blot, Incubation, Immunoprecipitation

Journal: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: GAS6 binds to its receptor, AXL, and p-AXL induce Schwann cell proliferation via MYC and Survivin signalling. GAS6 increases the expression level of CIP2A to form a p-AXL, p-DAPK and CIP2A protein complex. The presence of CIP2A inhibits the enzyme activity of PP2A and activates downstream ERK1/2 and AKT signals for proliferation.

Article Snippet: CIP2A cDNA (KIAA1524) was purchased from Origene (Rockville, MD).

Techniques: Expressing, Activity 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: Molecular medicine reports

Article Title: Expression and biological role of CIP2A in human astrocytoma.

doi: 10.3892/mmr.2013.1357

Figure Lengend Snippet: Figure 1. Expression of CIP2A in resected astrocytoma tissue. (A) Immunohistochemical staining of CIP2A protein in normal brain tissue was negative in glial cells and positive in neurons. (B) Negative CIP2A staining in pilocytic astrocytoma (grade I). (C) Negative CIP2A staining in grade II glioma. (D) Positive cytoplasmic CIP2A staining in grade III astro cytoma. (E) Positive cytoplasmic CIP2A staining in glioblastoma (grade IV). (F) Negative control. Magnification, x200. CIP2A, cancerous inhibitor of protein phosphatase 2A.

Article Snippet: Tissue sections were incubated with CIP2A rabbit polyclonal antibody (1:300 dilution; Novus Biologicals, LCC, Littleton, CO, USA) for 2 h at room temperature.

Techniques: Expressing, Immunohistochemical staining, Staining, Negative Control

Journal: Molecular medicine reports

Article Title: Expression and biological role of CIP2A in human astrocytoma.

doi: 10.3892/mmr.2013.1357

Figure Lengend Snippet: Figure 2. CIP2A expression and knockdown efficiency in astrocytoma cell lines. ��������������������������������������������������������������� (A) Endogenous expression of CIP2A was examined in five astrocy toma cell lines by western blot analysis and real‑time PCR. (B) western blot analysis and real‑time PCR revealed that siRNA treatment of CIP2A mark edly decreased CIP2A levels in A172 and U87 cells compared with negative control cells. CIP2A, cancerous inhibitor of protein phosphatase 2A.

Article Snippet: Tissue sections were incubated with CIP2A rabbit polyclonal antibody (1:300 dilution; Novus Biologicals, LCC, Littleton, CO, USA) for 2 h at room temperature.

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

Journal: Molecular medicine reports

Article Title: Expression and biological role of CIP2A in human astrocytoma.

doi: 10.3892/mmr.2013.1357

Figure Lengend Snippet: Figure 3 CIP2A knockdown inhibited cell proliferation in astrocytoma cell lines. (A) MTT assay revealed that CIP2A depletion inhibited cell proliferation. (B) Assessment of the clonogenic potential of CIP2A depleted glioma cells. The number of colonies formed by cells treated with CIP2A siRNA was reduced compared with control (P<0.05). (C) Anchorage‑independent colony formation assay revealed that CIP2A knockdown inhibited glioma cell growth in soft agar (P<0.05). (D) Annexin V/PI analysis revealed that CIP2A knockdown increased the level of apoptosis in astrocytoma cell lines. CIP2A, cancerous inhibitor of protein phosphatase 2A.

Article Snippet: Tissue sections were incubated with CIP2A rabbit polyclonal antibody (1:300 dilution; Novus Biologicals, LCC, Littleton, CO, USA) for 2 h at room temperature.

Techniques: Knockdown, MTT Assay, Control, Colony Assay

Journal: Molecular medicine reports

Article Title: Expression and biological role of CIP2A in human astrocytoma.

doi: 10.3892/mmr.2013.1357

Figure Lengend Snippet: Figure 4. CIP2A siRNA treatment downregulated c‑Myc, Bcl‑2, phos phor‑Akt expression and increased caspase‑3 cleavage in the astrocytoma cell line. Western blot analysis revealed that siRNA knockdown of CIP2A decreased the protein levels of c‑Myc, Bcl‑2 and phospho‑Akt in the two cell lines. Levels of cleaved caspase‑3 were increased following CIP2A depletion. CIP2A, cancerous inhibitor of protein phosphatase 2A.

Article Snippet: Tissue sections were incubated with CIP2A rabbit polyclonal antibody (1:300 dilution; Novus Biologicals, LCC, Littleton, CO, USA) for 2 h at room temperature.

Techniques: Expressing, Western Blot, Knockdown

Journal: Oncotarget

Article Title: Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.

doi: 10.18632/oncotarget.2941

Figure Lengend Snippet: Figure 2: Downregulation of CIP2A determined the effects of afatinib on p-AKT and apoptosis in NSCLC cells through PP2A activation. (A) Dose-dependent effects on CIP2A, p-AKT and AKT. (B) Effects of afatinib on PP2A activity. NSCLC cells were exposed to afatinib at the indicated concentrations for 24 h. Data are mean ± SD. n = 3 for each experiment. **, p < 0.01, vs. no afatinib. (C) Time-dependent effects of afatinib on CIP2A, p-AKT and apoptosis-related proteins in the sensitive H358 and resistant H460 cells. Cells were exposed to 10 μM afatinib for up to 48 hours. Immunoblots were scanned and quantitated to determine the ratio of the level of CIP2A to actin. Data are mean ± SD. n = 3 for the different time intervals.

Article Snippet: Gene knockdown using siRNA Smart-pool siRNA, including control (sc-37007), CIP2A and PP2A were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Activation Assay, Activity Assay, Western Blot

Journal: Oncotarget

Article Title: Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.

doi: 10.18632/oncotarget.2941

Figure Lengend Snippet: Figure 3: Validation of the CIP2A-PP2A-AKT pathway. (A) Left, ectopic expression of CIP2A (CIP2A-DDK) increased p-AKT and attenuated the effects of afatinib on apoptosis of H358 cells. H358 cells overexpressing CIP2A were treated with 10 μM afatinib for 24 h. Right, knockdown of CIP2A expression by siRNA increased the sensitivity to afatinib-induced apoptosis in H460 cells. Cells were transfected with either control or CIP2A siRNA for 48 h then exposed to 10 μM afatinib for 24 h. (B) Left, overexpression of CIP2A decreased PP2A activity in H358 cells. Right, downregulation of CIP2A by siRNA increased the activity of PP2A in H460 cells. (C) Silencing PP2Ac reduced the apoptotic effect of afatinib in H358 cells. Cells were transfected with either control or PP2Ac siRNA for 48 h then exposed to 10 μM afatinib for 24 h. (D) Left, okadaic acid (OA), a PP2A inhibitor, increased p-AKT and inhibited the effects of afatinib on apoptosis of H358 cells. Right, forskolin, a PP2A agonist, sensitized resistant H460 cells to afatinib. Data are mean ± SD. n = 3 for each condition. **, p < 0.01, vs. no afatinib. (E) Ectopic expression of AKT (AKT-DDK) attenuated the effects of afatinib on apoptosis of H358 cells. H358 cells overexpressing AKT were treated with 10 μM afatinib for 24 h.

Article Snippet: Gene knockdown using siRNA Smart-pool siRNA, including control (sc-37007), CIP2A and PP2A were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Biomarker Discovery, Expressing, Knockdown, Transfection, Control, Over Expression, Activity Assay

Journal: Oncotarget

Article Title: Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.

doi: 10.18632/oncotarget.2941

Figure Lengend Snippet: Figure 4: Elk-1 regulated CIP2A in NSCLC cells by afatinib. (A) Left, H358 cells were treated with 100 μg/ml cycloheximide (CHX) in the presence or absence of afatinib for the indicated length of time. Middle and Right, H358 and H460 cells treated with afatinib at 10 μM or 20 μM for the designated incubation time. Afatinib inhibited CIP2A mRNA in a dose- and time-dependent manner, especially in H358 cells. Data are mean ± SD. n = 3 for each time point. *, p < 0.05, **, p < 0.01, vs. no afatinib. (B) Effects of afatinib on CIP2A promoter activity. Left, H358 and H460 cells were co-transfected with CIP2A reporter constructs (-1 to -2000bp) and renilla luciferase vectors for 48 h then treated with 10 μM afatinib for an additional 24 h. Afatinib decreased CIP2A luciferase activity in H358 cells, but not in H460 cells. Right, H358 cells were transfected CIP2A reporter various lengths of constructs and renilla luciferase vector for 48 h and then treated with 2 μM or 10 μM afatinib for an additional 24 h. Cell lysates were prepared for analysis of luciferase activity. Data are mean ± SD. n = 3 for each condition. **, p < 0.01, vs. no afatinib. (C) Chromatin immunoprecipitation assays of the CIP2A promoter. H358 and H460 cells were treated with 2 μM or 10 μM afatinib for 24 h and processed for ChIP assay. Soluble chromatin was immunoprecipitated with specific Elk-1 or IgG (negative control) antibodies. Immunoprecipitates were subjected to PCR with primer pair specific to CIP2A promoter (-16 to -213 bp) and RPL30 (internal control). The gel shown is representative of three independent experiments. (D) H358 and H460 cells treated with afatinib at 2 μM or 10 μM for the indicated incubation times. Afatinib inhibited Elk-1 mRNA in a dose- and time-dependent manner, especially in H358 cells. Data are mean ± SD. n = 3 for each time point. **, p < 0.01, vs. no afatinib. (E) Ectopic expression of Elk1 (HA-Elk1) restored the effect of afatinib on CIP2A expression and protected the effect of afatinib-induced apoptosis in H358 cells using Western blotting and FACS. H358 cells overexpressing Elk-1 were treated with 10 μM afatinib for 24 h. Open arrow is endogenous of Elk1 and close arrow is exogenous of Elk1. (F) Knockdown of Elk-1 enhanced apoptosis in H460 cells by afatinib. Protein levels of Elk1 expressed in lower panel. Data are means ± SD. **, p < 0.01.

Article Snippet: Gene knockdown using siRNA Smart-pool siRNA, including control (sc-37007), CIP2A and PP2A were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Incubation, Activity Assay, Transfection, Construct, Luciferase, Plasmid Preparation, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Control, Expressing, Western Blot, Knockdown

Journal: Oncotarget

Article Title: Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.

doi: 10.18632/oncotarget.2941

Figure Lengend Snippet: Figure 5: In vivo effects of afatinib on xenografts mice. (A)(a) Left, effects of afatinib on tumor size in H358 xenografts. Afatinib inhibited the growth of H358 tumors by > 80%. Right, effects on body weight of H358 xenograft mice. (b) Left, effects of afatinib on tumor size in H460 xenograft mice. Right, effects on body weight of H460 xenograft mice. Mice were treated with vehicle, or oral afatinib at 20 mg/kg daily for 3 weeks (n = 8 each). Data are mean ± SD; *, p < 0.05. (B) Western blot analysis of CIP2A, p-AKT and AKT in H358 (Left) and H460 (Right) tumors. (C) The activities of PP2A in H358 (Left) and H460 (Right) tumors. mean ± SD, n = 8 for each condition.**p <0.01. (D) Representative immunohistochemical patterns of CIP2A and p-AKT in xenografts tumors.

Article Snippet: Gene knockdown using siRNA Smart-pool siRNA, including control (sc-37007), CIP2A and PP2A were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: In Vivo, Western Blot, Immunohistochemical staining

Journal: Oncotarget

Article Title: Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.

doi: 10.18632/oncotarget.2941

Figure Lengend Snippet: Figure 6: Cancerous inhibitor of protein phosphatase 2A (CIP2A) expression in 49 non-small cell lung cancer patients. (A) Strong, moderate, weak, and negative expressions of Elk-1, CIP2A, and p-AKT. (B) Elk-1 and CIP2A expression in the non-tumor part and tumor part; *, p < 0.05. (C) The correlation of Elk-1 and CIP2A, r= 0.773, p <0.001. (D) Kaplan-Meier estimates of progression-free survival in the 49 patients with NSCLC according to high or low CIP2A expression. (E) Schema of the molecular mechanism of afatinib on the Elk-1/CIP2A/PP2A/AKT pathway.

Article Snippet: Gene knockdown using siRNA Smart-pool siRNA, including control (sc-37007), CIP2A and PP2A were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Expressing

Journal: Cancer Medicine

Article Title: Cellular localization of CIP2A determines its prognostic impact in superficial spreading and nodular melanoma

doi: 10.1002/cam4.425

Figure Lengend Snippet: Expression of CIP2A according to subcellular localization

Article Snippet: Endogenous peroxidase was blocked using Dako blocking reagent for 5 min followed by incubation with rabbit polyclonal CIP2A antibody (NB100-74663; Novus Biologicals, Litleton, CO) diluted 1:200 (1.25 μ g IgG/mL) for 30 min.

Techniques: Expressing

Journal: Cancer Medicine

Article Title: Cellular localization of CIP2A determines its prognostic impact in superficial spreading and nodular melanoma

doi: 10.1002/cam4.425

Figure Lengend Snippet: (A) Kaplan–Meier survival curve showing the impact of high nuclear CIP2A expression and OS in superficial spreading melanoma ( n = 81). (B) Kaplan–Meier survival curve showing impact of high cytoplasmic CIP2A expression and RFS and (C) OS survival in nodular melanoma ( n = 51). Tables next to respective curves show number of patients at risk. CIP2A, cancerous inhibitor of protein phosphatase 2A; OS, overall survival; RFS, relapse-free survival.

Article Snippet: Endogenous peroxidase was blocked using Dako blocking reagent for 5 min followed by incubation with rabbit polyclonal CIP2A antibody (NB100-74663; Novus Biologicals, Litleton, CO) diluted 1:200 (1.25 μ g IgG/mL) for 30 min.

Techniques: Expressing

Journal: Cancer Medicine

Article Title: Cellular localization of CIP2A determines its prognostic impact in superficial spreading and nodular melanoma

doi: 10.1002/cam4.425

Figure Lengend Snippet: Associations between clinical parameters, signaling pathways, and CIP2A expression

Article Snippet: Endogenous peroxidase was blocked using Dako blocking reagent for 5 min followed by incubation with rabbit polyclonal CIP2A antibody (NB100-74663; Novus Biologicals, Litleton, CO) diluted 1:200 (1.25 μ g IgG/mL) for 30 min.

Techniques: Protein-Protein interactions, Expressing, Marker

Journal: Cancer Medicine

Article Title: Cellular localization of CIP2A determines its prognostic impact in superficial spreading and nodular melanoma

doi: 10.1002/cam4.425

Figure Lengend Snippet: Characterization of CIP2A expression in a panel of normal human melanocytes (NHM) and melanoma cell lines. (A) Immunoblot analysis and (B) quantification of protein expression levels of CIP2A (C) CIP2A mRNA expression measured by qRT-PCR. NHM1 line was used as calibrator. (D) Cytoplasmic and nuclear expression of CIP2A in normal melanocytes, primary and metastatic melanoma cell lines assessed by immunoblot analysis and (E) by immunohistochemical staining. CIP2A, cancerous inhibitor of protein phosphatase 2A.

Article Snippet: Endogenous peroxidase was blocked using Dako blocking reagent for 5 min followed by incubation with rabbit polyclonal CIP2A antibody (NB100-74663; Novus Biologicals, Litleton, CO) diluted 1:200 (1.25 μ g IgG/mL) for 30 min.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Cancer Medicine

Article Title: Cellular localization of CIP2A determines its prognostic impact in superficial spreading and nodular melanoma

doi: 10.1002/cam4.425

Figure Lengend Snippet: CIP2A downregulation reduces proliferation and induces apoptosis in melanoma cells. (A) The relative amount of viable cells measured by MTS assay 72 and 120 h post siRNA transfection. (B) Immunoblot analysis showing reduction in cancerous inhibitor of protein phosphatase 2A protein levels 48 h post siRNA transfection and effects on proliferation and apoptosis markers. The figure is representative of at last three independent biological experiments.

Article Snippet: Endogenous peroxidase was blocked using Dako blocking reagent for 5 min followed by incubation with rabbit polyclonal CIP2A antibody (NB100-74663; Novus Biologicals, Litleton, CO) diluted 1:200 (1.25 μ g IgG/mL) for 30 min.

Techniques: MTS Assay, Transfection, Western Blot

Journal: Oncotarget

Article Title: Lapatinib inhibits CIP2A/PP2A/p-Akt signaling and induces apoptosis in triple negative breast cancer cells.

doi: 10.18632/oncotarget.7035

Figure Lengend Snippet: Figure 1: Lapatinib exerts anti-proliferative and apoptotic-inducing effects in triple-negative breast cancer (TNBC) cells. A. Confirmation of HER2 and ER-alpha expression in TNBC cell lines (MDA-MB-231, MDA-MB-468, and HCC-1937). MCF-7 was used as a positive control for ER expression and SK-BR3 was used as a positive control for HER2 expression. B. Left, dose-escalation effects of lapatinib on cell viability; Middle and Right, effects of lapatinib (5 µM), anti-HER2 monoclonal antibody trastuzumab (40 µg/ml), or anti-EGFR monoclonal antibody cetuximab (20 µg/ml) on CIP2A in HER2-positive SK-BR3 cells. HER2, p-HER2, EGFR, p-EGFR, ERK, and p-ERK were assayed to confirm the target effects of these drugs. C. Dose-escalation effects of lapatinib on cell viability in TNBC cells. Cells were exposed to lapatinib at the indicated doses for 72 hours and cell viability was assessed by MTT assay. Points, mean (n = 3); bars, SD. D. Dose-and time-escalation effects of lapatinib on apoptosis in TNBC cell lines. Cells were exposed to lapatinib at the indicated doses for 24, 48, and 72 hours. Apoptotic cells were determined by flow cytometry (sub-G1 analysis of propidium iodide- stained cells). Columns, mean (n = 3); bars, SD.

Article Snippet: Generation of MDA-MB-468 cells with ectopically expressed CIP2A CIP2A cDNA (gene name KIAA1524) was purchased from Origene (RC219918; Rockville, MD, USA) and constructed into pCMV6 vector.

Techniques: Expressing, Positive Control, MTT Assay, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: Lapatinib inhibits CIP2A/PP2A/p-Akt signaling and induces apoptosis in triple negative breast cancer cells.

doi: 10.18632/oncotarget.7035

Figure Lengend Snippet: Figure 2: Lapatinib induces apoptosis in association with downregulation of CIP2A and p-Akt in TNBC cells. A. Dose- escalation effects of lapatinib on CIP2A, p-Akt, and caspase 3 cleavage. Cells were exposed to lapatinib at the indicated doses for 48 hours. B. time-dependent analysis of CIP2A, p-Akt, and caspase 3 cleavage. Cells were exposed to lapatinib (10 µM) for 24, 36 and 48 hours. Cell lysates were prepared and assayed for these molecules by western blotting. Data are representative of three independent experiments. Apoptotic cells were determined by flow cytometry (sub-G1 analysis of propidium iodide-stained cells).

Article Snippet: Generation of MDA-MB-468 cells with ectopically expressed CIP2A CIP2A cDNA (gene name KIAA1524) was purchased from Origene (RC219918; Rockville, MD, USA) and constructed into pCMV6 vector.

Techniques: Western Blot, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: Lapatinib inhibits CIP2A/PP2A/p-Akt signaling and induces apoptosis in triple negative breast cancer cells.

doi: 10.18632/oncotarget.7035

Figure Lengend Snippet: Figure 3: CIP2A/PP2A/p-Akt mediates lapatinib-induced apoptosis in TNBC cells. A. ectopic expression of myc-tagged CIP2A reduced the apoptotic effect of lapatinib in MDA-MB-468 cells. B. Analysis of PP2A activity in drug-treated TNBC cells. Cells were treated with DMSO or lapatinib at 10 μM or okadaic acid at 20 nM (as a negative control) or forskolin at 40 μM (as a positive control) for 24 hours. Cell lysates were assayed for PP2A activity. C. Pretreatment of PP2A inhibitor okadaic acid protected cells from lapatinib- induced apoptosis. Cells were pretreated with okadaic acid (20 nM) for 1 hour; then washed and treated with DMSO (control) or lapatinib (10 μM) for 24 hours. Cell lysates were separated and assayed for sub-G1 analysis and western blotting. D. Knockdown of PP2Ac reduced the effects of lapatinib on p-Akt and apoptosis. MDA-MB-468 cells were transfected with siRNA against PP2Ac (catalytic subunit) or control siRNA for 48h, after transfection cells were then treated with lapatinib 5μM for 24 h. Cell lysates were separated and assayed for sub-G1 analysis and western blotting.

Article Snippet: Generation of MDA-MB-468 cells with ectopically expressed CIP2A CIP2A cDNA (gene name KIAA1524) was purchased from Origene (RC219918; Rockville, MD, USA) and constructed into pCMV6 vector.

Techniques: Expressing, Activity Assay, Negative Control, Positive Control, Control, Western Blot, Knockdown, Transfection

Journal: Oncotarget

Article Title: Lapatinib inhibits CIP2A/PP2A/p-Akt signaling and induces apoptosis in triple negative breast cancer cells.

doi: 10.18632/oncotarget.7035

Figure Lengend Snippet: Figure 4: Lapatinib inhibits transcription of CIP2A. A. Effect of lapatinib on CIP2A protein degradation. Cells were treated with 100 μg/ml pan-translation inhibitor cyclohexamide (CHX) in the presence (right) or absence (left) of lapatinib (5 μM) for the indicated period of time, then the stability of CIP2A protein in whole-cell lysates was assessed by western blot. The addition of lapatinib did not significantly affect CIP2A degradation. B. Lapatinib inhibited CIP2A transcription. Cells were treated with lapatinib at the indicated doses for 24 hours, after which total RNA was isolated and CIP2A mRNA was assayed by real-time quantitative PCR. Columns, mean (n = 3); bars, SD. C. Luciferase reporter assay of CIP2A proximal promoter regions upon lapatinib treatment. MDA-MB-468 cells were transfected by Firefly luciferase reporter vectors carrying CIP2A promoters of different lengths as indicated, and Renilla vectors for 24 hours and then treated with 5 μM lapatinib or DMSO for 24 hours. Cell lysates were then assayed for dual luciferase activity as described in Materials and Methods. Columns, mean (n = 3); bars, SD; *, P < 0.05. D. lapatinib disturbed binding of Elk1 to the CIP2A promoter region. Left, lapatinib decreased Elk1 translocation from the cytosol to the nuclei. Nuclear and cytoplasmic extracts were prepared from MDA-MB-468 cells treated with lapatinib (5 µM) or DMSO for 24 hours. Cell lysates were western blotted for Elk1, and Ets1. Lamin B and Tubulin were used as a loading control. Right, chromatin immunoprecipitation assays of the CIP2A promoter were performed as described in Materials and Methods. Soluble chromatin was immunoprecipitated with Elk1, Ets1 or IgG (negative control) antibodies. Immunoprecipitates were subjected to PCR with primer pairs specific to the CIP2A promoter (-16 to -139 bp). The gel shown is representative of three independent experiments. Anti-RNA polymerase II antibody and GAPDH primers were used as a positive control for the assay technique and reagent integrity. E. Ectopic expression of Elk1 with pCMV-Elk1-GFP vector upregulated CIP2A expression, and suppressed lapatinib-induced CIP2A inhibition.

Article Snippet: Generation of MDA-MB-468 cells with ectopically expressed CIP2A CIP2A cDNA (gene name KIAA1524) was purchased from Origene (RC219918; Rockville, MD, USA) and constructed into pCMV6 vector.

Techniques: Western Blot, Isolation, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Transfection, Activity Assay, Binding Assay, Translocation Assay, Control, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Positive Control, Expressing, Plasmid Preparation, Inhibition

Journal: Oncotarget

Article Title: Lapatinib inhibits CIP2A/PP2A/p-Akt signaling and induces apoptosis in triple negative breast cancer cells.

doi: 10.18632/oncotarget.7035

Figure Lengend Snippet: Figure 5: In vivo effect of lapatinib on MDA-MB-468 xenograft nude mice. A. Lapatinib treatment decreased the size of MDA- MB-468 tumors. Points, mean (n = 6); bar, SE. *, P < 0.05. Mice were administered orally with 100 mg/kg body weight lapatinib three times weekly or vehicle (1× phosphate buffered saline), as described in Materials and Methods. B. tumor weight and C. PP2A activity in MDA-MB-468 xenografts treated with control or lapatinib. Columns, mean (n = 3); bars, SD; *, P < 0.05. D. Western blot analysis of the expression level of CIP2A, pAkt and Akt in MDA-MB-468 xenografts treated with control or lapatinib. E. Body weights of xenograft mice bearing tumors. Points, mean (n = 6); bar, SD. F. Hematoxylin and eosin (HE) stain, and immunohistochemical (IHC) staining for CIP2A, p-Akt and the proliferative index Ki-67 in the MDA-MB-468 xenografts treated with control or lapatinib. Power fields 200X. G. schema of the molecular mechanism of the action of lapatinib on the CIP2A/PP2A pathway. By indirectly inhibiting CIP2A transcription, lapatinib restores PP2A activity downregulating p-Akt and leading to subsequent cell apoptosis.

Article Snippet: Generation of MDA-MB-468 cells with ectopically expressed CIP2A CIP2A cDNA (gene name KIAA1524) was purchased from Origene (RC219918; Rockville, MD, USA) and constructed into pCMV6 vector.

Techniques: In Vivo, Saline, Activity Assay, Control, Western Blot, Expressing, H&E Stain, Immunohistochemical staining, Immunohistochemistry