Journal: Molecular Oncology

Article Title: Mislocalized cytoplasmic p27 activates PAK1‐mediated metastasis and is a prognostic factor in osteosarcoma

doi: 10.1002/1878-0261.12624

Figure Lengend Snippet: Correlation of clinical data with cytoplasmic p27 proportion scores in OS tissue microarray. (A) Bar graphs of the number of cases in different clinical categories by cytoplasmic p27 proportion score. Clinical categories from left to right are metastatic status at diagnosis, metastatic status at 3 years, metastatic status at 5 years, and histologic response to neoadjuvant chemotherapy. High cytoplasmic p27 score was significantly associated with development of metastatic disease within 3 years ( P = 0.018) or 5 years ( P = 0.014) of diagnosis. (B) Kaplan–Meier plots of EFS of OS patients. EFS was significantly associated with metastatic status at diagnosis ( P = 2.14E‐06), histologic response ( P = 1.49E‐04), and cytoplasmic p27 proportion score ( P = 0.0195) (top left, middle and right). EFS of OS cases without metastasis at diagnosis (NM, P = 0.027, bottom left), but not in cases with metastasis at diagnosis (ME, P = 0.567, bottom middle), was significantly associated with cytoplasmic p27 score. DSS of OS cases was also significantly associated with cytoplasmic p27 proportion score ( P = 0.045, bottom right). (C) Results of univariate and multivariate Cox proportional hazard model analyses for both EFS and DSS.

Article Snippet: Twenty‐three microlitre of 100 μg·mL −1 rabbit anti‐human p27 (D69C12) mAb (Cell Signaling) was added to the mixture and incubated at 4 °C overnight to form an immunocomplex.

Techniques: Microarray, Biomarker Discovery

Journal: Molecular Oncology

Article Title: Mislocalized cytoplasmic p27 activates PAK1‐mediated metastasis and is a prognostic factor in osteosarcoma

doi: 10.1002/1878-0261.12624

Figure Lengend Snippet: Characterization of the p27‐PAK1 interaction in OS cell lines. (A) Workflow of IP followed by mass spectrometry to identify cytoplasmic p27‐interacting proteins. (B) Results of p27 co‐IP with PAK1 in NES‐p27 cells, phosphosite mutants, and empty vector control. The pulldown assay was performed with rabbit p27 antibody (Ra‐p27), whereas PAK1 was detected by mouse PAK1 monoclonal antibody (Ma‐PAK1). Mouse monoclonal antibody p27 antibody (Ma‐p27) was used as an IP control, and total PAK1 expression was used as an input control. (C) Fluorescent images (20×) of cellular p27 and PAK1 showing p27 (EYFP, green) and PAK1 (immunofluorescence, red) proteins co‐localizing (merged, yellow) in the cytoplasm of NES‐p27 cells DAPI (blue) was used as nuclear counterstain. (D) Western blotting of phospho‐PAK1 in NES‐p27 cells, phosphosite mutants, and empty vector control. Total PAK1 protein was used as a loading control. (E) Images (left) and quantification (right) of phalloidin staining of F‐actin (stress fibers) in NES‐p27 cells, phosphosite mutants, and empty vector control (20×). For the quantification, experiments were repeated three times with ≥ 30 cells analyzed for each condition. (F) RAC1/CDC42 activity assays in NES‐p27 cells, phosphosite mutants, and empty vector control. RAC1 and CDC42 amounts of western blotting were used as input controls for RAC1/CDC42 activity assays. (G) Representative images (left) and quantification (right) of transwell migration assays of NES‐p27 and empty vector control cells treated with 1 µ m of the Group I PAK inhibitor (FRAX‐597) or the vehicle control (DMSO). Migrated cells were stained, counted, and averaged using imagej software (National Institute of Mental Health, Bethesda, MD, USA) in five random and independent microscopic fields (10×). Error bars and asterisks represent standard deviations and statistical significance (Student's t ‐test; * P < 0.05; ** P < 0.01; *** P < 0.001, ns, not significantly, respectively). The experiment was replicated three times with a similar result. (H) Western blots showing amounts of phospho‐PAK1 S144 and total PAK1 in the NES‐p27 mutant treated with FRAX‐597 or DMSO. (I) Representative images of phalloidin staining of actin stress fibers in NES‐p27 cells treated with FRAX‐597 or DMSO (40×).

Article Snippet: Twenty‐three microlitre of 100 μg·mL −1 rabbit anti‐human p27 (D69C12) mAb (Cell Signaling) was added to the mixture and incubated at 4 °C overnight to form an immunocomplex.

Techniques: Mass Spectrometry, Co-Immunoprecipitation Assay, Phospho-proteomics, Plasmid Preparation, Control, Expressing, Immunofluorescence, Western Blot, Staining, Activity Assay, Migration, Software, Mutagenesis

Journal: Molecular Oncology

Article Title: Mislocalized cytoplasmic p27 activates PAK1‐mediated metastasis and is a prognostic factor in osteosarcoma

doi: 10.1002/1878-0261.12624

Figure Lengend Snippet: Effects of PAK1 silencing on tumor cell migration and actin stress fiber formation in three distinct OS cell lines with p27 mislocalization. (A) Western blots of total PAK1 in PAK1‐shRNA (shRNA#1 and #2) mutants and scramble controls from NES‐p27, 143B, and U2OS cell lines. (B) Representative images of the transwell migration assays of the two PAK1‐shRNA mutants and the scramble controls in the three OS cell lines. (C) Quantification of the transwell migration assays shown in B. Migrated cells were stained, counted, and averaged using imagej software in five random and independent microscopic fields (10×). The experiment was replicated three times. (D) Representative images and quantification of phalloidin staining showing the amount and distribution of actin stress fibers in the two PAK1‐shRNA mutants and the scramble controls in the three OS cell lines (20×). In the quantification analyses, error bars and asterisks represent standard deviations and statistical significance (Student's t ‐test; * P < 0.05; ** P < 0.01; *** P < 0.001, ns, not significantly, respectively).

Article Snippet: Twenty‐three microlitre of 100 μg·mL −1 rabbit anti‐human p27 (D69C12) mAb (Cell Signaling) was added to the mixture and incubated at 4 °C overnight to form an immunocomplex.

Techniques: Migration, Western Blot, shRNA, Staining, Software

Journal: Molecular Oncology

Article Title: Mislocalized cytoplasmic p27 activates PAK1‐mediated metastasis and is a prognostic factor in osteosarcoma

doi: 10.1002/1878-0261.12624

Figure Lengend Snippet: Migration inhibitory effect of PAK1 silencing on non‐OS cell lines with p27 mislocalization. (A) Western blotting with subcellular fractionation of three non‐OS cancer cell lines. GAPDH and HDAC were used as nuclear and cytoplasmic protein controls, respectively (C, cytoplasmic fraction; N, nuclear fractions). (B) Western blotting of PAK1‐shRNA mutants and parental controls in the three cancer cell lines showing high efficiency of PAK1 knockdown. (C) Representative images and quantification of transwell migration assays showing migrated cells in the three PAK1‐shRNA mutants relative to their parental cells. Migrated cells were stained, counted, and averaged using imagej software in five random and independent microscopic fields (10×). Error bars represent standard deviations of the replicates, and asterisks denote statistical significance (Student's t ‐test; * P < 0.05; ** P < 0.01; *** P < 0.001, ns, not significantly, respectively). All experiments were replicated three times. (D) A model depicting how p27 mislocalization may lead to increased incidence of metastatic progression in the OS patients. Phosphorylated p27 in the cytoplasm interacts with PAK1, and the resulting protein–protein interaction activates PAK1 by protein phosphorylation. The PAK1 phosphorylation promotes actin polymerization and increases stress fiber formation in OS cells, leading to higher tumor cell migration and adhesion and, hence, metastasis.

Article Snippet: Twenty‐three microlitre of 100 μg·mL −1 rabbit anti‐human p27 (D69C12) mAb (Cell Signaling) was added to the mixture and incubated at 4 °C overnight to form an immunocomplex.

Techniques: Migration, Western Blot, Fractionation, shRNA, Knockdown, Staining, Software, Phospho-proteomics

Journal: Journal of Cancer

Article Title: The oncogenic potential of PRR11 gene in Tongue Squamous Cell Carcinoma cells

doi: 10.7150/jca.29265

Figure Lengend Snippet: PRR11 regulated the expressions of cell cycle-related proteins. (A) The protein levels of cell cycle-related proteins of PRR11-overexpressing and PRR11-silencing TSCC cells at 72 h after transfection were determined by Western blot. (B) The expression of P21 and P27 mRNA was determined using real-time PCR.

Article Snippet: Primary antibodies included rabbit anti-human PRR11 polyclonal antibody (1:250, NOVUS, USA), rabbit anti-human Cyclin A2 polyclonal antibody (1:1000, Santa Cruz, USA), rabbit anti-human Cyclin B1 monoclonal antibody, (1:1000, Cell Signaling, USA), rabbit anti-human Cyclin D1 monoclonal antibody (1:1000, Cell Signaling), rabbit anti-human monoclonal antibody p21 (1:1000, Cell Signaling), rabbit anti-human monoclonal antibody p27 (1:1000, Cell Signaling), rabbit anti-human monoclonal antibody β-actin antibody (1:1000, Abcam, USA) were used in this study.

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

Journal: Journal of biological engineering

Article Title: A facile in vitro platform to study cancer cell dormancy under hypoxic microenvironments using CoCl 2 .

doi: 10.1186/s13036-018-0106-7

Figure Lengend Snippet: Fig. 8 MCF-7 and MDA-MB-231 cells exhibit differential gene and protein expression profiles in response to CoCl2 treatment. a Fold change in mRNA expression of CDKN1A, CDKN1B, CDK2, CDK4, CCNA2, CCND1, CCNE1, and MYC by qRT-PCR after 72 h of CoCl2 treatment relative to untreated control (* P < 0.05 compared to untreated control). b Western blot analysis of HIF1α, p21, p27 and β-actin (control) expression in MCF-7 and MDA-MB- 231 cells after 72 h of CoCl2 treatment in 2D and 3D (pHEMA-coated plate) cultures compared to untreated control. c Relative protein expression of p21 normalized to β-actin, with results represented as mean ± SD of three independent experiments (* P < 0.05 compared to untreated control)

Article Snippet: For p21, p27, p38 MAPK, pp38 MAPK, ERK(1/2), and pERK(1/2), the membranes were incubated with rabbit anti-human p21 primary antibody (12D1), rabbit anti-human p27 primary antibody (D69C12), rabbit anti-human p38 MAPK primary antibody, rabbit anti-human pp38 MAPK primary antibody, rabbit anti-human ERK(1/2) primary antibody and rabbit anti-human pERK(1/2) primary antibody (1:1000, Cell Signaling Technology) overnight at 4 °C.

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