Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: High p21 expression correlates with poor survival in breast cancer patients . A-C , The relationship of higher p21 expression and breast cancer outcome was assessed by Kaplan-Meier survival analysis. The survival rates were analyzed using three different and separate splits of the patients, based on p21 expression levels (A, median; B, upper quartile; C, lower quartile). For each split, the patients were then divided into high and low groups with respect to p21 expression. Overall survival (left panels) and distant metastasis-free survival (right panels) correlate with p21 gene expression. Number of breast cancer patients at risk with higher expression (red) and lower expression (black) of p21 at the indicated time points.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Gene Expression

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: Silencing p21 prevents breast tumor local invasion in vivo and cancer cell migration and invasion . A , Total lysates from parental and shRNA p21 SCP2 cells were analyzed by immunoblotting for the protein levels of p21 and β-tubulin. B , Parental and shRNA p21 SCP2 cells were injected into the mammary glands of four- to six-week-old female Balb/c nude mice. The size of mammary tumor was measured from two sets of mice (eight per group; error bars indicate SEM). C , Representative photographs show hematoxylin and eosin staining of the mammary gland (tumor and fat pad) of 12- to 15-month-old mice. D and E , Transwell cell migration (left panel) and GFR-Matrigel invasion assay (right panel) of parental and shRNA p21 SCP2 cells were performed. Graphs show total migrated and invaded cell number counted by Image J (error bars indicate SEM; n = 3 independent experiments). Student's unpaired t -test was used to compare parental vs. shRNA p21 SCP2 groups. Differences were considered significant at * P < 0.05.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: In Vivo, Migration, shRNA, Western Blot, Injection, Staining, Invasion Assay

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: TGFβ induces p21 expression in migratory and invasive human breast cancer cells . A , Real-time PCR was performed to measure the mRNA level of p21 gene (error bars indicate SD; n = 3 independent experiments) for the indicated cell lines. B , Cells were treated with or without 5 ng/ml TGFβ for the indicated times. Total cell lysates were analyzed for p21 and β-tubulin protein levels by Western blotting. C , Total cell lysates were analyzed for c-myc, p15 and β-tubulin protein levels by Western blotting. D , SCP25 cells were pretreated with 10 µM TGFβ type I receptor (TβRI) inhibitor (SB431542) or vehicle (DMSO) for 30 minutes and then stimulated with TGFβ. Total cell lysates were analyzed for p21 and β-tubulin protein levels by Western blotting. E , MDA cells were transfected with 40 nM Scrambled (Scr), Smad2 or Smad3 siRNAs in response to TGFβ. Total cell lysates were analyzed for Smad2/3, p21 and β-tubulin protein levels by Western blotting.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

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

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 expression is required for TGFβ-mediated cell migration . A , Representative images of phase contrast and wound mask of indicated cell lines stimulated with TGFβ in scratch/wound healing assay. The initial wound mask (black) and wound closure (grey) were measured using the Essen Instruments Scratch Wound Module. B , The time course of cell migration for the indicated cell lines was quantified using the relative wound density metrics at two-hour time intervals (error bars indicate SEM; n = 3 independent experiments). C , SCP2 cells were transfected with Scr or p21 siRNAs and then stimulated with or without TGFβ for 24 hrs. Total cell lysates were analyzed for p21 and β-tubulin by Western blotting. D , Representative images of phase contrast (top panels) and wound mask (bottom panels) of transfected SCP2 cells with the indicated siRNAs in scratch/wound healing assay. E , The time course of transfected SCP2 cell migration was quantified using the relative wound density metrics at two-hour time intervals (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Migration, Wound Healing Assay, Transfection, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 expression is required for TGFβ-mediated cell invasion . A , SCP2 cells were transfected with the indicated siRNAs and cultured in the presence or absence of TGFβ. GFR-Matrigel coated Transwell invasion assay was performed and images of the invading cells were photographed. B , Total cell number was counted by Image J and fold induction was quantified (error bars indicate SD; n = 3 independent experiments). C , SCP2 cells were transfected with a Scr or p21 siRNA as well as a flag-tagged p21 cDNA in the presence or the absence of TGFβ. p21 protein levels were then analyzed by Western blotting. D , Cell invasion was assessed using the Transwell Invasion assay. The number of invaded cells was counted by Image J (error bars indicate SD; n = 3 independent experiments). E , Cell invasion of transfected SUM159 was assessed using the Transwell Invasion assay. F , Total cell number was counted by Image J and fold induction was quantified (error bars indicate SD; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Transfection, Cell Culture, Transwell Invasion Assay, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 interacts with Smad3 and modulates TGFβ-induced transcriptional activity . A , HEK293 and SCP2 cells were co-transfected with myc-Smad2, myc-Smad3 and flag-p21. Transfected cells were stimulated TGFβ for 8 hrs. Cell lysates were immunoprecipitated with an anti-flag antibody and analyzed by immunoblotting using Smad2/3 and p21 antibodies. B , Transfected MDA cells were immunoblotting by phospho-Smad3 (p-Smad3), Smad2/3 and β-tubulin antibodies in response to TGFβ for 30 minutes. C , SCP2 cells were co-transfected with either mock, Scr siRNA, p21 siRNA or flag-tagged p21 construct (p21 cDNA) and SBE promoter construct (CAGA12-luc). Transfected cells were stimulated with or without TGFβ for 16 hrs. Luciferase activity of CAGA12-luc was measured and normalized to β-galactosidase (error bars indicate SEM; n = 3 independent experiments). D and E , SUM159 and SCP2 cells were treated with or without TGFβ for the indicated times. The mRNA levels of indicated genes were then analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Activity Assay, Transfection, Immunoprecipitation, Western Blot, Construct, Luciferase, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 regulates TGFβ-induced downstream genes involved in cell invasion . A , SUM159 cells were transfected with Scr or p21 siRNA as well as p21 cDNA. Cells were then treated with or without TGFβ and the mRNA levels of indicated genes were analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments). B , SUM159 cells transfected with Scr siRNA and p21 siRNA were treated with TGFβ for the indicated times. The mRNA level of TGFBI gene was measured by real-time PCR (error bars indicate SEM; n = 3 independent experiments). C , SCP2 cells were transfected with the indicated siRNAs. Cell invasion was assessed using the Transwell Invasion assay. D , Total cell number was counted by Image J and number of invaded cells was quantified (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Transwell Invasion Assay

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21/p/CAF regulates TGFβ transcriptional activity and Smad3 occupancy on SBE . A , SCP2 and SUM159 cells were treated with TGFβ. Cell lysates were analyzed by co-immunoprecipitation using specific antibodies, as indicated. B , HEK293 cells were co-transfected with myc-Smad2, myc-Smad3 and flag-p21 with or without p/CAF siRNA. Transfected cells were stimulated with TGFβ for eight hours. Cell lysates were immunoprecipitated with an anti-flag antibody and analyzed by immunoblotting using Smad2/3 and flag antibodies. C and D , SUM159 cells were transfected with Scr or p/CAF siRNAs as well as a flag-tagged p21 cDNA, treated with or without TGFβ. The mRNA levels of indicated genes were analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments). E , SCP2 cells transfected with Scr and p/CAF siRNA were stimulated with or without TGFβ. Cell invasion was quantified by relative TGFβ fold induction (error bars indicate SEM; n = 3 independent experiments). F , Transfected SCP2 cells were subjected to immunoblotting p/CAF and β-tubulin. G , HEK293 cells were co-transfected with myc-Smad3, myc-Smad2 and p/CAF. Immunoprecipitated Smad2/3 using an anti-myc antibody was subjected to Western blotting. H , DNA precipitation (DNA IP) was performed using biotinylated control and 4× CAGA SBE oligonucleotides, following by streptavidin precipitation. Western blotting of Smad3 and p/CAF is shown. I , SCP2 cells were transfected with p21 or p/CAF siRNAs. Samples were subjected to DNA IP and immunoblotting of Smad3. J , Transfected SCP2 cells were stimulated with or without TGFβ for 16 hrs. Luciferase activity of CAGA12-luc was measured and normalized to β-galactosidase (error bars indicated SEM; n =3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Activity Assay, Immunoprecipitation, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Control, Luciferase

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: High expression of p/CAF/p21/pSmad3 is associated with lymph node positivity . A , Representative immunohistochemistry images of pSmad3, p21 and p/CAF in breast cancer tissue microarray samples. B , Overall pSmad3, p21 and p/CAF immunohistochemistry staining intensity between lymph node negative (LN-) and positive (LN+) tissues. C , Percentage of case distribution according to immunoreactivity of pSmad3, p21 and p/CAF in tumor cells, the score for negative (0), low (1 to 2) and high (3 to 4).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Staining

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: Model of pro-invasive function for the cell cycle regulator p21 in human breast cancer . The role of p21 as both a direct transcriptional target of TGFβ and a co-stimulatory factor of p/CAF/Smad3 in regulation of pro-invasive genes in triple negative breast cancer cells.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques:

Journal: Journal of Veterinary Research

Article Title: Immunoreactivity of p21, MMP-1 and CB2 receptor proteins in cutaneous canine mast cell tumours: an association with the three-tier grading system

doi: 10.2478/jvetres-2023-0066

Figure Lengend Snippet: Immunohistochemistry with primary anti-p21 antibody with positive reaction visible as brown staining. A – weak nuclear reaction in a grade 1 mast cell tumour (MCT); B – moderate nuclear reaction in a grade 2 MCT; C – strong nuclear reaction in a grade 3 MCT

Article Snippet: The primary antibodies used in this study, all canine-specific, were as follows: anti-CB2R (Cat. No. TA317640; OriGene Technologies, Rockville, MD, USA), anti-p21 (Cat. No. M7202; clone SX118, Agilent Dako, Santa Clara, CA, USA) and anti-MMP-1 (Cat. No. ABIN2777120; antibodies-online, Aachen, Germany), diluted 1 : 200, 1 : 50 and 1 : 500, respectively.

Techniques: Immunohistochemistry, Staining

Journal: Journal of Veterinary Research

Article Title: Immunoreactivity of p21, MMP-1 and CB2 receptor proteins in cutaneous canine mast cell tumours: an association with the three-tier grading system

doi: 10.2478/jvetres-2023-0066

Figure Lengend Snippet: Frequency of immunoreactivity for p21 protein in grade 1, 2 and 3 mast cell tumours (MCTs). * – P-value < 0.05; **_ P-value < 0.01; *** P-value < 0.001 compared to grade 1 MCTs; (−) – no reaction; (+) – weak reaction; (++) – moderate reaction; (+++) – strong reaction

Article Snippet: The primary antibodies used in this study, all canine-specific, were as follows: anti-CB2R (Cat. No. TA317640; OriGene Technologies, Rockville, MD, USA), anti-p21 (Cat. No. M7202; clone SX118, Agilent Dako, Santa Clara, CA, USA) and anti-MMP-1 (Cat. No. ABIN2777120; antibodies-online, Aachen, Germany), diluted 1 : 200, 1 : 50 and 1 : 500, respectively.

Techniques:

Journal: British Journal of Cancer

Article Title: Impact of differential cyclin D1 expression and localisation in prostate cancer

doi: 10.1038/sj.bjc.6603615

Figure Lengend Snippet: Contribution of p21 Cip1 expression in prostate carcinoma. Scatter plots characterising nuclear p21 Cip1 (%) in a subset of patient tumours (PT1, n =16), which had been immunostained for cyclin D1 and Ki-67. ( A ) A trend between increased nuclear p21 Cip1 and Gleason score was observed. ( B ) A positive correlation by linear regression was identified between increased nuclear p21 Cip1 and Ki-67 (%) ( r 2 =0.72). ( C ) Tumours with more nuclear D1 appear to have more nuclear p21 Cip1 . Average values for panels A and C are described in the Results section.

Article Snippet: Immunohistochemistry for p21 Cip1 (1 : 1000, SC-397; Santa Cruz, CA, USA) and AR (1 : 5000, SC-816; Santa Cruz, CA, USA) was performed using a Vectastain Elite ABC rabbit staining kit according to manufacturer specifications (Vector Laboratories, Inc., Burlingame, CA, USA).

Techniques: Expressing

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A) CAMK2N1, AR and p21 protein levels were determined by Western blot in LNCaP cells with stably knockdown of CAMK2N1. LNCaP cells were treated with 10 nM R1881 for 10 hrs. (B-F) CAMK2N1, AR, PSA, TMPRSS2 and p21 mRNA levels were determined by qRT-PCR in LNCaP cells with stably knockdown of CAMK2N1. LNCaP cells were treated with 10 nM R1881 for 10 hrs. (G-J) Androgen-responsive luciferase reporter genes (PSA-Luc, MMTV-Luc) were assessed for AR activity. LNCaP cells with CAMK2N1 overexpression or knockdown were treated with R1881 for 24 hrs. (K) CHIP analysis of AR for PSA promoter region in LNCaP cells with stable knockdown of CAMK2N1. LNCaP cells were treated with R1881 or vehicle for 10 hrs. CHIP assay was performed using an anti-AR antibody.

Article Snippet: Tumor tissues were stained with primary antibody including Ki67 (RM-9106-S1, Thermo), Bcl-2 (SC-7382, Santa cruz), Bax (SC-7480, Santa cruz), p21 (SC-6246, Santa cruz).

Techniques: Western Blot, Stable Transfection, Knockdown, Quantitative RT-PCR, Luciferase, Activity Assay, Over Expression

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A) Expression levels of pAKT ser473 , AKT, AR, Bcl-2, BAX, and p21 were determined by Western blot in LNCaP cells with stable CAMK2N1 knockdown. (B) Expression levels of pAKT ser473 , AKT and AR were determined by Western blot in LNCaP cells with stable CAMK2N1 overexpression. (C-D) CAMK2N1 knockdown cells treated with 10 nM R1881 and/or 20μM KN-93 inhibitor. PSA-Luc was assessed for AR activity. CAMKIIβ and AR protein levels were determined by Western blot. (E-F) LNCaP cells transiently transfected with CAMK2N1 and/or CAMKIIβ while treated with 10 nM R1881. PSA-Luc was assessed for AR activity. CAMK2N1 and CAMKIIβ protein levels were determined by Western blot. (G-H) CAMK2N1 knockdown cells treated with 10 nM R1881 and/or 20 μM AKT VIII. PSA-Luc was assessed for AR activity. pAKT ser473 , AKT and AR β protein levels were determined by Western blot. (I-J) LNCaP cells transiently transfected with CAMK2N1 and/or m-AKT. PSA-Luc was assessed for AR activity. CAMK2N1 and CAMKIIβ protein levels were determined by Western blot.

Article Snippet: Tumor tissues were stained with primary antibody including Ki67 (RM-9106-S1, Thermo), Bcl-2 (SC-7382, Santa cruz), Bax (SC-7480, Santa cruz), p21 (SC-6246, Santa cruz).

Techniques: Expressing, Western Blot, Knockdown, Over Expression, Activity Assay, Transfection

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A) Expression levels of pAKT ser473 , AKT, AR, Bcl-2, BAX, and p21 were determined by Western blot in C4-2 cells with stable CAMK2N1 knockdown. (B) Expression levels of pAKT ser473 , AR Bcl-2, BAX, and p21 were determined by Western blot in C4-2 cells with stable CAMK2N1 overexpression. (C) C4-2 cells with stable overexpression of CAMK2N1 and AR knockdown. Cells were analyzed for cell proliferation by MTT assay. CAMK2N1 and AR protein levels were determined by Western blot. (D) C4-2 cells with stable overexpression of CAMK2N1 followed by treatment with or without Casodex (10 μM) were analyzed for cell proliferation by MTT. (E) C4-2 cells with stable knockdown of CAMK2N1 were analyzed for cell cycle by flow cytometry. (F) C4-2 cells with stable overexpression of CAMK2N1 and AR knockdown. Cells were analyzed for cell apoptosis by Annexin V staining.

Article Snippet: Tumor tissues were stained with primary antibody including Ki67 (RM-9106-S1, Thermo), Bcl-2 (SC-7382, Santa cruz), Bax (SC-7480, Santa cruz), p21 (SC-6246, Santa cruz).

Techniques: Expressing, Western Blot, Knockdown, Over Expression, MTT Assay, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A-C) C4-2 tumors with stable CAMK2N1 knockdown were injected into nude mice. Tumor size was measured every 5 days. The data was shown as mean ± SEM for N > 6 separate tumors for each group. (A) Images of tumors dissected from the mice. (B) The tumor size (mm 3 ) versus days of post injection. (C) Tumor was weighted after resection at the end of experiment. (D) mRNA levels of AR, PSA, P21, BAX and BCL2 were determined by qRT-PCR in tumors. (E) IHC staining detected the protein expression of CAMK2N1, AR, p21, Ki67, pAKT ser473 and BCL2 in C4-2 tumor tissues derived from mice. Data for quantified IHC was shown as mean ± SEM for N = 4 tumors in each group.

Article Snippet: Tumor tissues were stained with primary antibody including Ki67 (RM-9106-S1, Thermo), Bcl-2 (SC-7382, Santa cruz), Bax (SC-7480, Santa cruz), p21 (SC-6246, Santa cruz).

Techniques: Knockdown, Injection, Quantitative RT-PCR, Immunohistochemistry, Expressing, Derivative Assay

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Primers for amplifying PAK6 and GSK3β.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Sequencing

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Expression levels of PAK6 in cervical carcinoma and paracarcinoma tissues.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Expressing

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Association between PAK6 expression levels and clinicopathological parameters in cervical cancer.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Expressing

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: PAK6 expression in cervical cancer tissues and in C33A and HeLa cells. (A) Immunohistochemistry was used to analyze the expression levels of PAK6 in cervical cancer tissues. Scale bars, 200 µm. (B) Expression levels of PAK6 in C33A and HeLa cells were analyzed using western blotting. (C) Semi-quantification of the PAK6 protein expression levels presented in part (B) using ImageJ software. **P<0.01 vs. HeLa cells. PAK6, p21-activated kinase 6.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Expressing, Immunohistochemistry, Western Blot, Software

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Effect of the knockdown of PAK6 expression levels on the proliferation, migration and invasion of HeLa cells. (A) PAK6 mRNA expression levels were analyzed in stably shPAK6-transfected HeLa cells. (B) PAK6 protein expression levels were analyzed in stably shPAK6-transfected HeLa cells using western blotting. (C) Semi-quantification of PAK6 expression levels presented in part (B). (D) Cell Counting Kit-8 assays and (E) colony formation assays were used to analyze the proliferative rate of shPAK6-transfected HeLa cells. (F) Semi-quantification of the number of colonies formed from part (E). (G) Cell migration and invasion were determined in stably shPAK6-transfected HeLa cells, (magnification ×200). (H) Semi-quantification of the number of invasive cells from part (G). (I) Semi-quantification of the number of migratory cells from part (G). **P<0.01 vs. shPAK6 NC. PAK6, p21-activated kinase 6; sh, short hairpin RNA; NC, negative control.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Knockdown, Expressing, Migration, Stable Transfection, Transfection, Western Blot, Cell Counting, shRNA, Negative Control

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Effects of the overexpression of PAK6 on the proliferation, migration and invasion of HeLa cells. (A) PAK6 mRNA expression levels in stable PAK6 overexpressing HeLa cells were analyzed. (B) PAK6 protein expression levels were analyzed in stable PAK6 overexpressing HeLa cells using western blotting. (C) Semi-quantification of PAK6 expression levels presented in part (B). (D) Cell Counting Kit-8 assays and (E) colony formation assays were used to analyze the proliferative rate of stable PAK6 overexpressing HeLa cells. (F) Semi-quantification of the number of colonies formed from part (E). (G) Cell migration and invasion were determined in stable overexpressing PAK6 HeLa cells, (magnification ×200). (H) Semi-quantification of the number of invasive cells from part (G). (I) Semi-quantification of the migratory cell number from part (G). *P<0.05, **P<0.01 vs. PAK6 NC. PAK6, p21-activated kinase 6; NC, negative control.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Over Expression, Migration, Expressing, Western Blot, Cell Counting, Negative Control

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Effect of PAK6 knockdown or overexpression on the Wnt/β-catenin signaling pathway in HeLa cells. Western blotting was used to analyze the expression levels of (A) β-catenin, p-β-catenin, GSK3β and p-GSK3β, and (B) E-cadherin and Cyclin D1 in stably shPAK6-transfected HeLa cells. (C) Semi-quantification of the expression levels of proteins in parts (A) and (B). *P<0.05, **P<0.01 vs. shPAK6 NC. Western blotting was used to analyze the expression levels of (D) β-catenin, p-β-catenin, GSK3β and p-GSK3β, and (E) E-cadherin and cyclin D1 in stable PAK6 overexpressing HeLa cells. (F) Semi-quantification of the expression levels of proteins in parts (D) and (E). *P<0.05, **P<0.01 vs. PAK6 NC. (G) Immunofluorescence was used to demonstrate the co-localization of PAK6 and GSK3β. Scale bars, 10 µm. (H) Co-IP was used to analyze the interaction between PAK6 and GSK3β. PAK6, p21-activated kinase 6; sh, short hairpin RNA; NC, negative control; p-, phosphorylated; GSK3β, glycogen synthase kinase 3β; IP, immunoprecipitation.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Knockdown, Over Expression, Western Blot, Expressing, Stable Transfection, Transfection, Immunofluorescence, Co-Immunoprecipitation Assay, shRNA, Negative Control, Immunoprecipitation

Journal: bioRxiv

Article Title: Canonical Wnt signalling is activated during BEC-to-hepatocyte conversion in vivo and modulates liver epithelial cell plasticity in hepatic organoids

doi: 10.1101/2020.11.09.374462

Figure Lengend Snippet: (A) Treatment time for the flow cytometry isolation of BECs (tdTom+ Epcam+ cells) from MCD-injured livers following administration of either AAV8.p21 or AAV8.null viral particles via tail injection. (B) Representative images of MCD-injured livers following 6-day recovery. Dash line highlights the presence of HNF4a tdTomBEC-derived hepatocytes. (n=4 animals) (C and D) RT-qPCR gene expression analysis of tdTom+ Epcam+ cells isolated at various time points from the AAV8.null (non-transdifferentiation, panel C) and the AAV8.p21 (BEC-to-hepatocyte transdifferentiation, panel D) MCD injury models. (n=4 animals per condition). (E) Axin2 RNAscope analysis of serial sections of AAV8.p21 MCD-injured livers at day recovery 6 (top). Immunohistochemistry for tdTom labels cells of ductal origin (bottom). Arrowheads point to BECs in ducts. Asterisks point to BECs Axin2+ cells showing an invasive phenotype. (n=3 animals).

Article Snippet: Animals received a single dose of AAV8.TBG.null or AAV8.TBG.p21 (5*10^12 units/ml) (Addgene) intravenously allowed 1 week wash-out period on normal diet.

Techniques: Flow Cytometry, Isolation, Injection, Derivative Assay, Quantitative RT-PCR, Gene Expression, RNAscope, Immunohistochemistry

Journal: bioRxiv

Article Title: Canonical Wnt signalling is activated during BEC-to-hepatocyte conversion in vivo and modulates liver epithelial cell plasticity in hepatic organoids

doi: 10.1101/2020.11.09.374462

Figure Lengend Snippet: Scheme integrating the dynamics of BEC-to-hepatocyte transdifferentation and the expression patterns of Axin2, Lrp6 and Lgr5 obtained by RT-qPCR analysis of BECs (tdTom+ Epcam+) isolated at different time points during the recovery period of the p21 MCD mouse model. BEC-to-hepatocyte conversion occurs predominantly between day 3 and day 7 of the recovery period . Lrp6 gene expression (blue line) gradually declines between day 3 and day 9. By contrast, Lgr5 gene expression (green line) gradually increases between day 3 and day 9 of the recovery period. Expression of the Wnt/β-catenin target gene Axin2 (magenta line) is significantly increased at day 6 of recovery. Sections represented with dash lines represent predicted gene expression levels.

Article Snippet: Animals received a single dose of AAV8.TBG.null or AAV8.TBG.p21 (5*10^12 units/ml) (Addgene) intravenously allowed 1 week wash-out period on normal diet.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Gene Expression

Journal: Journal of Cancer

Article Title: P21-activated kinase 7 (PAK7) interacts with and activates Wnt/β-catenin signaling pathway in breast cancer

doi: 10.7150/jca.24934

Figure Lengend Snippet: PAK7 is significantly upregulated in human breast cancer tissues and cells. A, PAK7 mRNA expression was upregulated in breast cancer tissues (15/20) than paired normal breast tissues which was analyzed by RT-qPCR (x-axis represent 20 pairs of tissue samples). B, PAK7 protein expression was increased in breast cancer, which was detected in 110 cases of human breast cancer tissue microarray by immunohistochemistry. C, The protein levels of PAK7 in poor differentiation of breast cancer tissues were higher than that in well differentiation of breast cancer tissues. D, The protein levels of PAK7 were higher in stage II than that in stage I, according to TNM staging of breast cancer. E, PAK7 mRNA and protein expression were detected by RT-qPCR and western blotting in normal breast cell line MCF-10A and breast cancer cell lines MCF-7 and MDA-MB-231. Values represent the mean ± SD from three independent measurements. The brown staining indicates expression levels of PAK7 protein by immunohistochemistry. *P < 0.05, ***P < 0.001.

Article Snippet: The following primary antibodies were used for western blotting and analyses: PAK7 (Proteintech, USA), flag (Proteintech, USA), GFP (Proteintech, USA), GSK3β (Proteintech, USA), p-GSK3β (Cell Signaling Technology, USA), β-catenin (Proteintech, USA), p-β-catenin (Cell Signaling Technology, USA), c-myc (Proteintech, USA), cyclin D1 (Proteintech, USA) and GAPDH (Proteintech, USA),and the goat anti-rabbit IgG (Proteintech, USA) and goat anti-mouse IgG (Proteintech, USA).

Techniques: Expressing, Quantitative RT-PCR, Microarray, Immunohistochemistry, Western Blot, Staining

Journal: Journal of Cancer

Article Title: P21-activated kinase 7 (PAK7) interacts with and activates Wnt/β-catenin signaling pathway in breast cancer

doi: 10.7150/jca.24934

Figure Lengend Snippet: PAK7 promotes proliferation of breast cancer cells, and inhibits cell apoptosis. A, The cell were transfected with flag-PAK7 overexpression plasmid or flag-NC negative control vector, and small interfering RNA of PAK7 (siR-PAK7) or negative control (siR-NC) were detected by RT-qPCR and western blotting in MCF-7 and MDA-MB-231 cells. B, PAK7 overexpression enhances the proliferation of MCF-7 cells which was detected by the CCK-8 assay. C, PAK7 knockdown inhibits the proliferation of MDA-MB-231 cells which was detected by the CCK-8 assay. D, PAK7 overexpression enhances the proliferation of MCF-7 cells which was detected by the colon formation assay. E, PAK7 knockdown inhibits the proliferation of MDA-MB-231 cells which was detected by the colon formation assay. F, PAK7 overexpression increases the portion of MCF-7 cells in S and G2/M phase which was detected by flow cytometry. G, PAK7 knockdown increases the portion of MDA-MB-231 cells in G1 phase, and decreases the portion of cells in S and G2/M phase which was detected by flow cytometry. H, PAK7 overexpression inhibits early apoptosis and late apoptosis of MCF-7 cells which was detected by flow cytometry. I, PAK7 knockdown promotes early apoptosis and late apoptosis of MDA-MB-231 cells which was detected by flow cytometry. Values represent the mean ± SD from three independent measurements. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The following primary antibodies were used for western blotting and analyses: PAK7 (Proteintech, USA), flag (Proteintech, USA), GFP (Proteintech, USA), GSK3β (Proteintech, USA), p-GSK3β (Cell Signaling Technology, USA), β-catenin (Proteintech, USA), p-β-catenin (Cell Signaling Technology, USA), c-myc (Proteintech, USA), cyclin D1 (Proteintech, USA) and GAPDH (Proteintech, USA),and the goat anti-rabbit IgG (Proteintech, USA) and goat anti-mouse IgG (Proteintech, USA).

Techniques: Transfection, Over Expression, Plasmid Preparation, Negative Control, Small Interfering RNA, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Knockdown, Tube Formation Assay, Flow Cytometry

Journal: Journal of Cancer

Article Title: P21-activated kinase 7 (PAK7) interacts with and activates Wnt/β-catenin signaling pathway in breast cancer

doi: 10.7150/jca.24934

Figure Lengend Snippet: PAK7 significantly promote the migration of breast cancer cells. A, PAK7 overexpression enhances the migration ability of MCF-7 cells which was detected by Wound healing assay. B, PAK7 knockdown suppresses the migration ability of MDA-MB-231 which was detected by Wound healing assay. C, PAK7 overexpression enhances the migration ability of MCF-7 cells which was detected by Transwell assay. D, PAK7 knockdown suppresses the migration ability of MDA-MB-231 which was detected by Transwell assay. Values represent the mean ± SD from three independent measurements. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The following primary antibodies were used for western blotting and analyses: PAK7 (Proteintech, USA), flag (Proteintech, USA), GFP (Proteintech, USA), GSK3β (Proteintech, USA), p-GSK3β (Cell Signaling Technology, USA), β-catenin (Proteintech, USA), p-β-catenin (Cell Signaling Technology, USA), c-myc (Proteintech, USA), cyclin D1 (Proteintech, USA) and GAPDH (Proteintech, USA),and the goat anti-rabbit IgG (Proteintech, USA) and goat anti-mouse IgG (Proteintech, USA).

Techniques: Migration, Over Expression, Wound Healing Assay, Knockdown, Transwell Assay

Journal: Journal of Cancer

Article Title: P21-activated kinase 7 (PAK7) interacts with and activates Wnt/β-catenin signaling pathway in breast cancer

doi: 10.7150/jca.24934

Figure Lengend Snippet: PAK7 activates Wnt/β-catenin signaling pathway in breast cancer cells. A, The effect of transfecting with flag-PAK7 overexpression plasmid or flag-NC negative control vector on the protein levels of flag-PAK7, β-catenin, p-β-catenin (S33/S37/T41), GSK3β, p-GSK3β(S9), c-myc, cyclin D1 and β-catenin (nucleus), c-myc (nucleus) in MCF-7 cells. B, The effect of transfecting with small interfering RNA of PAK7 (siR-PAK7) or negative control (siR-NC) vector on the protein levels of flag-PAK7, β-catenin, p-β-catenin (S33/S37/T41), GSK3β, p-GSK3β(S9), c-myc, cyclin D1 and β-catenin (nucleus), c-myc (nucleus) in MDA-MB-231 cells. C, PAK7 overexpression increases the activity of Wnt/β-catenin signaling pathway which was detected by TOP/FOP flash assay in MCF-7 cells. D, PAK7 knockdown inhibits the activity of Wnt/β-catenin signaling pathway which was detected by TOP/FOP flash assay in MDA-MB-231 cells. E, The expression of PAK7 was positively correlated with β-catenin (CTNNB1), c-myc (MYC) expression which was analyzed in GEPIA database by bioinformatics methods. Values represent the mean ± SD from three independent measurements. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The following primary antibodies were used for western blotting and analyses: PAK7 (Proteintech, USA), flag (Proteintech, USA), GFP (Proteintech, USA), GSK3β (Proteintech, USA), p-GSK3β (Cell Signaling Technology, USA), β-catenin (Proteintech, USA), p-β-catenin (Cell Signaling Technology, USA), c-myc (Proteintech, USA), cyclin D1 (Proteintech, USA) and GAPDH (Proteintech, USA),and the goat anti-rabbit IgG (Proteintech, USA) and goat anti-mouse IgG (Proteintech, USA).

Techniques: Over Expression, Plasmid Preparation, Negative Control, Small Interfering RNA, Activity Assay, Knockdown, Expressing

Journal: Journal of Cancer

Article Title: P21-activated kinase 7 (PAK7) interacts with and activates Wnt/β-catenin signaling pathway in breast cancer

doi: 10.7150/jca.24934

Figure Lengend Snippet: PAK7 inhibits the degradation of β-catenin via phosphorylating GSK3β. A, PAK7 knockdown suppressed p-GSK3β (S9) expression. pEGFP-β-catenin was transfected into HEK293T cells along with siR-PAK7 or negative control. Forty-eight hours after transfection, cells were harvested for western blotting analysis to detect the expression of PAK7, GFP-β-catenin, GSK3β, p-GSK3β. B, PAK7 knockdown inhibited β-catenin mediated transcriptional activity of TCF. HEK293T cells were cotransfected with siR-PAK7 or siR-NC, pEGFP-β-catenin, TOP flash (TOP) or FOP flash (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. C, Inhibiting proteasomes degradation by MG132 reverses the effect of PAK7 knockdown on β-catenin degradation. HEK293T cells were cotransfected with pEGFP-β-catenin and siR-PAK7 or siR-NC. Forty-four hours after transfection, the cells were treated with 30μM MG132. Cells were harvested 4 hours later and subjected to western blotting analysis to detect the expression of GFP-β-catenin and PAK7. D, Inhibiting GSK3β activity by Licl reduced β-catenin expression inhibition by PAK7 knockdown. siR-PAK7 or siR-NC was transfected into MDA-MB-231 cells. 36 hours after transfection, the cells were treated with 30 mM Licl for 12 hours and then harvested for western blotting analysis to detect the expression of GFP-β-catenin, p-GSK3β (S9) and PAK7. E, siR-PAK7 or siR-NC was transfected into MDA-MB-231 cells. 36 hours after transfection, the cells were treated with 30 mM Licl for 12 hours and harvested for luciferase activity assay. F, MDA-MB-231 cells were transfected with flag-NC and flag-PAK7, treated with Licl for 48 hours and harvested for western blot assay.

Article Snippet: The following primary antibodies were used for western blotting and analyses: PAK7 (Proteintech, USA), flag (Proteintech, USA), GFP (Proteintech, USA), GSK3β (Proteintech, USA), p-GSK3β (Cell Signaling Technology, USA), β-catenin (Proteintech, USA), p-β-catenin (Cell Signaling Technology, USA), c-myc (Proteintech, USA), cyclin D1 (Proteintech, USA) and GAPDH (Proteintech, USA),and the goat anti-rabbit IgG (Proteintech, USA) and goat anti-mouse IgG (Proteintech, USA).

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

Journal: Journal of Cancer

Article Title: P21-activated kinase 7 (PAK7) interacts with and activates Wnt/β-catenin signaling pathway in breast cancer

doi: 10.7150/jca.24934

Figure Lengend Snippet: PAK7 interacts with β-catenin and GSK3β. A, PAK7 was binding to β-catenin and GSK3β by immunoprecipitation. β-catenin (GFP tagged) was cotransfected with PAK7 (flag tagged) or empty vector into HEK293T cells. Immunoprecipitation was performed with flag antibody and GFP antibody, respectively. β-catenin, GSK3β and PAK7 were analyzed with a GFP antibody, GSK3β antibody, and flag antibody, respectively. B, PAK7 and β-catenin were co-localized in the cell cytoplasm and nucleus. PAK7 (GFP tagged) and pCherry-β-catenin (Cherry tagged) were cotransfected into HEK293T cells. Co-localization (yellow fluorescence) of PAK7 (green fluorescence) and β-catenin (red fluorescence) was detected in the cytoplasm and nucleus. C, Working model for the regulation of β-catenin degradation by PAK7 via phosphorylating GSK3β in Wnt/β-catenin signaling pathway.

Article Snippet: The following primary antibodies were used for western blotting and analyses: PAK7 (Proteintech, USA), flag (Proteintech, USA), GFP (Proteintech, USA), GSK3β (Proteintech, USA), p-GSK3β (Cell Signaling Technology, USA), β-catenin (Proteintech, USA), p-β-catenin (Cell Signaling Technology, USA), c-myc (Proteintech, USA), cyclin D1 (Proteintech, USA) and GAPDH (Proteintech, USA),and the goat anti-rabbit IgG (Proteintech, USA) and goat anti-mouse IgG (Proteintech, USA).

Techniques: Binding Assay, Immunoprecipitation, Plasmid Preparation, Fluorescence

Journal: Journal of Cancer

Article Title: P21-activated kinase 7 (PAK7) interacts with and activates Wnt/β-catenin signaling pathway in breast cancer

doi: 10.7150/jca.24934

Figure Lengend Snippet: PAK7 expression and clinicopathologic characteristics of TMA

Article Snippet: The following primary antibodies were used for western blotting and analyses: PAK7 (Proteintech, USA), flag (Proteintech, USA), GFP (Proteintech, USA), GSK3β (Proteintech, USA), p-GSK3β (Cell Signaling Technology, USA), β-catenin (Proteintech, USA), p-β-catenin (Cell Signaling Technology, USA), c-myc (Proteintech, USA), cyclin D1 (Proteintech, USA) and GAPDH (Proteintech, USA),and the goat anti-rabbit IgG (Proteintech, USA) and goat anti-mouse IgG (Proteintech, USA).

Techniques: Expressing

Journal: Scientific Reports

Article Title: LINC01088 inhibits tumorigenesis of ovarian epithelial cells by targeting miR-24-1- 5 p

doi: 10.1038/s41598-018-21164-9

Figure Lengend Snippet: PAK4 is the potential target of miR-24-1-5p . ( A ) Prediction of binding sites between miR-24-1-5p and PAK4 . Gene Blasting was performed and two potential binding sites between miR-24-1-5p and PAK4 were found. ( B ) PAK4 in primary epithelial ovarian tumors. IHC staining was performed to measure PAK4 expression in (ii) normal ovarian epithelial tissue, (iii) benign epithelial ovarian tumor tissue, while (i) was shown as the negative control (100X, bar = 50 um). The higher magnification (400X, bar = 50 um) was shown in the upper right corner, correspondingly. ( C ) Validating the interaction between miR-24-1-5p and PAK4 . The relative luciferase activity was notably decreased in A2780 cells co-transfected with pcDNA6.2-GW/EmGFP-miR-24-1-5p , PRL-TK and pMIR-PAK4 3′UTR reporter plasmids. miR-LacZ was taken as the control. Data are represented as mean +/−SD, **means P < 0.01 vs control ( ANOVA ). ( D ) Detection of PAK4 in miR-24-1-5p -expressing cells. Western blot was conducted to examine the protein expression of PAK4 in A2780 cells stably transfected with pcDNA6.2-GW/ EmGFP-miR-24-1-5p or control plasmids (cropped; full length blots can be found in Supplementary Fig. ).

Article Snippet: Then the membrane was incubated with rabbit anti-human primary antibody PAK4 (1:500–1:1000, Abcam, UK), mouse Anti-Flag Tag (1:1000–1:10 000, Proteintech, USA) and mouse anti-human primary antibody GAPDH (1:5000, KangChen Bio-tech Inc. China) at 4 °C overnight, respectively.

Techniques: Binding Assay, Immunohistochemistry, Expressing, Negative Control, Luciferase, Activity Assay, Transfection, Control, Western Blot, Stable Transfection

Journal: Scientific Reports

Article Title: LINC01088 inhibits tumorigenesis of ovarian epithelial cells by targeting miR-24-1- 5 p

doi: 10.1038/s41598-018-21164-9

Figure Lengend Snippet: PAK4 inhibits cell proliferation. A2780 cells transfected with pCMV6-Entry-PAK4 or control plasmids for 48 h were harvested to perform the experiments as follows. ( A ) Detection of PAK4 in stable PAK4-expressing cells. Western blot was conducted to examine the protein expression of PAK4 in two groups (cropped; full length blots can be found in Supplementary Fig. ). ( B ) Determination of proliferation. MTS assay was performed to determine cell proliferation. Data are represented as mean +/− SD, ** means P < 0.01 vs control group ( ANOVA ). ( C ) Determination of migration. Scratch assay was performed to determine cell migration. Data are represented as mean +/− SD, ** means P < 0.01 vs control group ( ANOVA ).

Article Snippet: Then the membrane was incubated with rabbit anti-human primary antibody PAK4 (1:500–1:1000, Abcam, UK), mouse Anti-Flag Tag (1:1000–1:10 000, Proteintech, USA) and mouse anti-human primary antibody GAPDH (1:5000, KangChen Bio-tech Inc. China) at 4 °C overnight, respectively.

Techniques: Transfection, Control, Expressing, Western Blot, MTS Assay, Migration, Wound Healing Assay

Journal: Scientific Reports

Article Title: LINC01088 inhibits tumorigenesis of ovarian epithelial cells by targeting miR-24-1- 5 p

doi: 10.1038/s41598-018-21164-9

Figure Lengend Snippet: Schematic representation of the interaction among LINC01088 , miR-24-1-5p and PAK4 .

Article Snippet: Then the membrane was incubated with rabbit anti-human primary antibody PAK4 (1:500–1:1000, Abcam, UK), mouse Anti-Flag Tag (1:1000–1:10 000, Proteintech, USA) and mouse anti-human primary antibody GAPDH (1:5000, KangChen Bio-tech Inc. China) at 4 °C overnight, respectively.

Techniques: