Journal: BMC Cancer

Article Title: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an independent prognostic marker in wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy

doi: 10.1186/s12885-015-1300-3

Figure Lengend Snippet: Model of CIP2A involvement in the EGFR-RAS signaling pathways. ETS1 mediates CIP2A overexpression in human cancers with increased EGFR-MEK-ERK pathway activity. A positive feedback loop of CIP2A and MEK/ERK signaling pathways is shown.

Article Snippet: CIP2A expression was assessed by IHC using monoclonal antibodies to CIP2A (NB100-74663, 1:1200; Novus Biologicals, Littleton, CO, USA).

Techniques: Protein-Protein interactions, Over Expression, Activity Assay

Journal: BMC Cancer

Article Title: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an independent prognostic marker in wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy

doi: 10.1186/s12885-015-1300-3

Figure Lengend Snippet: Association between clinicopathological parameters and CIP2A expression in patients after colorectal liver metastasectomy

Article Snippet: CIP2A expression was assessed by IHC using monoclonal antibodies to CIP2A (NB100-74663, 1:1200; Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Significance Assay, Biomarker Discovery, Mutagenesis

Journal: BMC Cancer

Article Title: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an independent prognostic marker in wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy

doi: 10.1186/s12885-015-1300-3

Figure Lengend Snippet: Immunohistochemical (IHC analysis of CIP2A expression in patients with colorectal cancer. Representative examples of CIP2A expression: (a) strong expression in colorectal liver metastasis, (b) weak expression in colorectal liver metastasis, (c) staining in paired colon cancer, (d) colorectal metastasis tissues, and (e) H-score in paired colon cancer and liver metastasis samples. CIP2A was not consistently overexpressed in colon cancer compared with colorectal liver metastasis in paired tissue specimens.

Article Snippet: CIP2A expression was assessed by IHC using monoclonal antibodies to CIP2A (NB100-74663, 1:1200; Novus Biologicals, Littleton, CO, USA).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: BMC Cancer

Article Title: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an independent prognostic marker in wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy

doi: 10.1186/s12885-015-1300-3

Figure Lengend Snippet: Prognostic factors for overall survival according to univariate and multivariate analyses in patients with both wild-type and mutant KRAS metastatic colorectal cancer after colorectal liver metastasectomy

Article Snippet: CIP2A expression was assessed by IHC using monoclonal antibodies to CIP2A (NB100-74663, 1:1200; Novus Biologicals, Littleton, CO, USA).

Techniques: Mutagenesis, Significance Assay, Biomarker Discovery, Over Expression

Journal: BMC Cancer

Article Title: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an independent prognostic marker in wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy

doi: 10.1186/s12885-015-1300-3

Figure Lengend Snippet: Kaplan–Meier survival plot of overall survival (OS) by KRAS genotype. (a) OS was significantly worse in patients with wild type KRAS and strong CIP2A expression, compared with patients with wild type KRAS and weak CIP2A expression ( P = 0.035). (b) No difference in OS associated with CIP2A expression was observed in mutant KRAS patients ( P = 0.759).

Article Snippet: CIP2A expression was assessed by IHC using monoclonal antibodies to CIP2A (NB100-74663, 1:1200; Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Mutagenesis

Journal: BMC Cancer

Article Title: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an independent prognostic marker in wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy

doi: 10.1186/s12885-015-1300-3

Figure Lengend Snippet: Prognostic factors for overall survival according to univariate and multivariate analyses in patients with wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy

Article Snippet: CIP2A expression was assessed by IHC using monoclonal antibodies to CIP2A (NB100-74663, 1:1200; Novus Biologicals, Littleton, CO, USA).

Techniques: Significance Assay, Biomarker Discovery

Journal: BMC Cancer

Article Title: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an independent prognostic marker in wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy

doi: 10.1186/s12885-015-1300-3

Figure Lengend Snippet: Interaction between CIP2A, KRAS genotype and proliferation in the Caco-2 KRAS wild-type cell line. The result of immunoblot and proliferation assay is shown in (a) and (b) , respectively. Column 1 vs. 2: CIP2A knockdown by shCIP2A resulted in decreased CIP2A, KRAS, and pERK expression as well as decreased proliferation; Column 1 vs. 3: KRAS overexpression by pCMV6-KRAS G12D resulted in increased KRAS, CIP2A, and pERK expression, as well as increased proliferation; Column 3 vs. 4: in Caco-2 cells with KRAS overexpression by pCMV6-KRAS G12D, knockdown of CIP2A by shCIP2A resulted in decreased CIP2A and KRAS expression. However, it did not cause significantly decreased pERK expression or decreased proliferation (*P < 0.05).

Article Snippet: CIP2A expression was assessed by IHC using monoclonal antibodies to CIP2A (NB100-74663, 1:1200; Novus Biologicals, Littleton, CO, USA).

Techniques: Western Blot, Proliferation Assay, Knockdown, Expressing, Over Expression

Journal: BMC Cancer

Article Title: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an independent prognostic marker in wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy

doi: 10.1186/s12885-015-1300-3

Figure Lengend Snippet: Silencing of CIP2A in Caco-2 cells leads to decreased resistance to cetuximab. Immunoblot analysis of CIP2A expression in control (shLuc) and CIP2A knockdown (shCIP2A) cells.

Article Snippet: CIP2A expression was assessed by IHC using monoclonal antibodies to CIP2A (NB100-74663, 1:1200; Novus Biologicals, Littleton, CO, USA).

Techniques: Western Blot, Expressing, Control, Knockdown

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: mRNA-seq of rat tissues. To explore the related molecular mechanism of bronchiolitis obliterans, high-throughput analysis was conducted using the lung tissues of rats. (A) DEGs in DA group rats relative to the control are shown using a volcano plot. DEGs were defined as an absolute value of log2FC >1.5 and adjusted-P<0.001. (B) Quantitative PCR was carried out to verify the results of mRNA-seq. Four DEGs were randomly selected for verification. (C) Gene Ontology analysis revealed that apoptosis, inflammation, fibrosis, EMT and epithelium-associated items were enriched by DEGs. (D) Venn diagram shows that 47 DEGs commonly existed in the six datasets. (E) Heatmap of 47 DEGs demonstrated that most genes were functional proteins. (F) CIP2A was located on chromosome 11 and was increased in DA-treated rats. n=6. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; DEGs, differentially expressed genes; EMT, epithelial-mesenchymal transition; FC, fold change; mRNA-seq, mRNA-sequencing; SLC1A6, solute carrier family 1 member 6; ERN2, endoplasmic reticulum to nucleus signaling 2; RNASE2, ribonuclease A family member 2; TPSB2, tryptase β2.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: High Throughput Screening Assay, Control, Real-time Polymerase Chain Reaction, Functional Assay, Sequencing

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: CIP2A expression is increased in DA group rats. (A) Quantitative PCR and western blotting, and (B) immunohistochemistry (scale bar: 50 µm) were used to detect the increased expression of CIP2A in samples from the DA group, which was initially determined by mRNA-sequencing. n=6. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Sequencing

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting CIP2A attenuates the injuries induced by DA in the bronchi and lungs of rats. To evaluate the effect of CIP2A inhibition on bronchiolitis obliterans, Eth, a CIP2A inhibitor, was injected subcutaneously into DA group rats. (A) Animal experimental timeline. Representative (B) H&E and (C) Masson's trichrome-stained lung slices (scale bar: 200 µm). Quantitative analysis of severity is shown. (D) Bronchoalveolar lavage fluid total cell count, and macrophage, neutrophil, lymphocyte and eosinophil counts in the control rats, DA-exposed rats and Eth-treated DA-exposed rats. (E) Western blotting and (F) immunohistochemistry were used to measure the protein expression levels of CIP2A in lung tissues. n=6. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; Eth, ethoxysanguinarine; H&E, hematoxylin and eosin.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: Inhibition, Injection, Staining, Cell Counting, Control, Western Blot, Immunohistochemistry, Expressing

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting cell proliferation regulating inhibitor of protein phosphatase 2A weakens the epithelial-mesenchymal transition and inflammation induced by DA in the bronchi and lungs of rats. (A) E-cadherin and (B) Vimentin levels were detected by immunofluorescence (scale bar: 50 µm). (C) Western blotting was used to measure the protein expression levels of α-SMA, fibronectin, Snail and iNOS. (D) Enzyme-linked immunosorbent assay kits were used to analyze the levels of IL-1β, IL-6 and TNF-α. n=6. α-SMA, α-smooth muscle actin; CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; IL, interleukin; iNOS, inducible NO synthase; TNF-α, tumor necrosis factor-α.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: Immunofluorescence, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting CIP2A reduces damage caused by DA in human primary bronchial epithelial cells. To explore the cellular mechanisms, an in vitro cellular model of bronchiolitis obliterans was employed. (A) Eth (6 µM) showed the best inhibitory effect on CIP2A expression. (B) Western blotting was used to measure the protein expression levels of α-SMA, fibronectin, Snail and iNOS. (C) Enzyme-linked immunosorbent assay kits were used to analyze the levels of IL-1β, IL-6 and TNF-α. (D) E-cadherin and (E) Vimentin levels were detected by immunofluorescence (scale bar: 50 µm). n=3. α-SMA, α-smooth muscle actin; CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; Eth, ethoxysanguinarine; IL, interleukin; iNOS, inducible NO synthase; TNF-α, tumor necrosis factor-α.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: In Vitro, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting CIP2A blocks the activation of NF-κB signaling in HPBECs. The expression of CIP2A, and phosphorylation of IKBα and expression of p65 were assessed by western blotting. n=3. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; HPBECs, human primary bronchial epithelial cells; IκBα, inhibitor of NF-κB α; NF-κB, nuclear factor-κB; p-, phosphorylated.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: Activation Assay, Expressing, Western Blot

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: CIP2A promotes BO by activating the NF-κB pathway. BO is caused by DA and mediated by CIP2A. CIP2A deficiency inhibits fibrosis, inflammation and EMT in BO models through suppressing the NF-κB pathway. Solid and dashed arrows both indicate promotion. The solid arrow reveals the molecular mechanism through which the dashed arrow operates. BO, bronchiolitis obliterans; DA, diacetyl; CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; EMT, epithelial-mesenchymal transition; NF-κB, nuclear factor-κB.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques:

Journal: Journal of the College of Physicians and Surgeons--Pakistan : JCPSP

Article Title: Relationship Between CIP2A and Endometrium Cancer.

doi: 10.29271/jcpsp.2020.04.373

Figure Lengend Snippet: Figure 1: Representative images of immunohistochemical staining for endometrial carcinoma and atypical endometrial hyperplasia. (A, B) Strong CIP2A expression in the endometrial carcinoma (x200). (C,D) Strong CIP2A expression in the atypical endometrial hyperplasia (x200).

Article Snippet: Primary antibodies CIP2A (Novus Biologicals - 1/40 dilution), HER2 (Cell Marque / RabMab - 1/50 dilution), Ki-67 (Dako Flex - 1/100 dilution) and P53 (Dako Flex - 1/100 dilution) were dropped manually and incubated for 30 minutes duration.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Journal of the College of Physicians and Surgeons--Pakistan : JCPSP

Article Title: Relationship Between CIP2A and Endometrium Cancer.

doi: 10.29271/jcpsp.2020.04.373

Figure Lengend Snippet: Figure 2: (A) Expression levels of CIP2A mRNA in normal endometrium and endometrial carcinoma. (B) The relationship between CIP2A mRNA expres- sion and endometrial groups. (C) The relationship between CIP2A mRNA expression and CIP2A protein expression. NE: Normal endometrium, EH: Endometrial hyperplasia, AEH: Atypical endometrial hyperplasia, EC: Endometrial carcinoma.

Article Snippet: Primary antibodies CIP2A (Novus Biologicals - 1/40 dilution), HER2 (Cell Marque / RabMab - 1/50 dilution), Ki-67 (Dako Flex - 1/100 dilution) and P53 (Dako Flex - 1/100 dilution) were dropped manually and incubated for 30 minutes duration.

Techniques: Expressing

Journal: Journal of the College of Physicians and Surgeons--Pakistan : JCPSP

Article Title: Relationship Between CIP2A and Endometrium Cancer.

doi: 10.29271/jcpsp.2020.04.373

Figure Lengend Snippet: Figure 3: Relationship of CIP2A mRNA expression with clinicopathological parameters.

Article Snippet: Primary antibodies CIP2A (Novus Biologicals - 1/40 dilution), HER2 (Cell Marque / RabMab - 1/50 dilution), Ki-67 (Dako Flex - 1/100 dilution) and P53 (Dako Flex - 1/100 dilution) were dropped manually and incubated for 30 minutes duration.

Techniques: Expressing

Journal: Science Advances

Article Title: Mapping the biogenesis of forward programmed megakaryocytes from induced pluripotent stem cells

doi: 10.1126/sciadv.abj8618

Figure Lengend Snippet: Antibodies. FITC, fluorescein isothiocyanate; PE, phycoerythrin; HLA, human leukocyte antigen; IgG, immunoglobulin G.

Article Snippet: CIP2A , APCCy7 , Novus Biologicals , NB110-59722APCCY7 , 1:20.

Techniques: Control

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: 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: International journal of colorectal disease

Article Title: CIP2A regulates MYC translation (via its 5'UTR) in colorectal cancer.

doi: 10.1007/s00384-020-03772-y

Figure Lengend Snippet: Fig. 1 Depletion of CIP2A regulates MYC protein and cell proliferation in CRC cell lines. a Western blot analysis of CIP2A and MYC protein expression in HCT116, SW480, and LS174t cells transfected with CIP2A or CTR siRNA for 72 h. Data are representative of three independent experiments. b RT-QPCR analysis of CIP2A and MYC mRNA expres- sion in HCT116 72 h after transfection with siRNA targeting CIP2A (mean of three independent biological experiments, error bars represent +/−SD). c Crystal violet staining of HCT116 cells stably infected with CIP2A or CTR shRNA after 7 days in culture. d Cell number of HCT116

Article Snippet: Western blots were probed with antibodies against CIP2A (A301-454A; Bethyl Laboratories), MYC (C33, #42; Santa Cruz; or Y69 # ab32072 Abcam), βactin (AC-15/A5441; Sigma), vinculin (V9131; Sigma), and HA-tag (HA-Tag; C29F4 #3724S cell signaling).

Techniques: Western Blot, Expressing, Transfection, Quantitative RT-PCR, Staining, Stable Transfection, Infection, shRNA

Journal: International journal of colorectal disease

Article Title: CIP2A regulates MYC translation (via its 5'UTR) in colorectal cancer.

doi: 10.1007/s00384-020-03772-y

Figure Lengend Snippet: Fig. 2 Depletion of CIP2A does not alter MYC protein stability in CRC cells. Immunoblots documenting MYC protein stability. Cells were incubated with cycloheximide (50 μg/ml) for the indicated time. a HCT116 left panel, immunoblot; right panel, quantification of MYC to vinculin ratio. b SW480 left panel, immunoblot; right panel,

Article Snippet: Western blots were probed with antibodies against CIP2A (A301-454A; Bethyl Laboratories), MYC (C33, #42; Santa Cruz; or Y69 # ab32072 Abcam), βactin (AC-15/A5441; Sigma), vinculin (V9131; Sigma), and HA-tag (HA-Tag; C29F4 #3724S cell signaling).

Techniques: Western Blot, Incubation