Journal: Journal of Cancer

Article Title: EXOSC5 promotes proliferation of gastric cancer through regulating AKT/STAT3 signaling pathways.

doi: 10.7150/jca.69166

Figure Lengend Snippet: Figure 4. EXOSC5 Knockdown led to G1 Arrest in GC. (A,B) Flow cytometry showing the percentages of EXOSC5 knockdown cells and control cells at different cell cycle phase. (C) Cell cycle related proteins (P21, P27, Cyclin D1) in EXOSC5 knockdown and EXOSC5 overexpression cells by Western blot. Data are shown as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The primary antibodies are as following: EXOSC5 (Proteintech, 15627-1-AP); p21 (Proteintech, 10355-1-AP); p27 (Proteintech, 25614-1-AP); cyclin D1 (CST, 2922S); p-AKT (CST, 9271S); AKT (CST, 9272S); p-STAT3 (CST, 9145); AKT (CST, 9272S).

Techniques: Knockdown, Flow Cytometry, Control, Over Expression, Western Blot, Standard Deviation

Journal: Journal of Cancer

Article Title: EXOSC5 promotes proliferation of gastric cancer through regulating AKT/STAT3 signaling pathways.

doi: 10.7150/jca.69166

Figure Lengend Snippet: Figure 5. EXOSC5 promote GC growth by AKT and STAT3 activation. (A) KEGG pathway analysis of the genes significantly associated with the EXOSC5 expression in GC from cBioPortal and Coexpedia. (B) The protein expression levels of p-AKT, AKT, p-STAT3 and STAT3 in GC cell lines after EXOSC5 silencing or overexpressing. (C, D) Proliferation ability of MKN45 cells with EXOSC5 overexpression was determined by CCK8 and colony formation assay after treatment with MK-2206 (10 uM), S31-201 (10 uM) and DMSO. (E) The protein expression levels of p-AKT, AKT, p-STAT3, STAT3 and cell cycle related proteins (cyclin D1, p21 and p27) in MKN45 cells with EXOSC5 overexpression after treatment with MK-2206, S31-201 and DMSO. Data are shown as the mean ± standard deviation. ***P < 0.001.

Article Snippet: The primary antibodies are as following: EXOSC5 (Proteintech, 15627-1-AP); p21 (Proteintech, 10355-1-AP); p27 (Proteintech, 25614-1-AP); cyclin D1 (CST, 2922S); p-AKT (CST, 9271S); AKT (CST, 9272S); p-STAT3 (CST, 9145); AKT (CST, 9272S).

Techniques: Activation Assay, Expressing, Over Expression, Colony Assay, Standard Deviation

Journal: Journal of Cancer

Article Title: EXOSC5 promotes proliferation of gastric cancer through regulating AKT/STAT3 signaling pathways.

doi: 10.7150/jca.69166

Figure Lengend Snippet: Figure 6. EXOSC5 promote GC growth by AKT and STAT3 activation in GC organoid and vivo. (A) Hematoxylin-eosin staining (H&E) of GC organoid (400x). (B) Immunohistochemistry (IHC) of GC organoid after EXOSC5 silencing. (C) The protein expression levels of p-AKT, AKT, p-STAT3, STAT3 and cell cycle related proteins (cyclin D1, p21 and p27) in GC organoid after EXOSC5 silencing or overexpressing. (D, E) The growth of organoid model with EXOSC5 knockdown or overexpression was assessed every 7 days. (F) Representative images of subcutaneous tumors in nude mice injected HGC27 cells transferred with shRNA. (G, H) Tumor volumes were measured by growth curve every 5 days and final weights of tumor were measured on the terminal days. (I) EXOSC5 staining in xenografted HGC27 tumors silencing EXOSC5. Data are shown as the mean ± standard deviation. **P < 0.01, ***P < 0.001.

Article Snippet: The primary antibodies are as following: EXOSC5 (Proteintech, 15627-1-AP); p21 (Proteintech, 10355-1-AP); p27 (Proteintech, 25614-1-AP); cyclin D1 (CST, 2922S); p-AKT (CST, 9271S); AKT (CST, 9272S); p-STAT3 (CST, 9145); AKT (CST, 9272S).

Techniques: Activation Assay, Staining, Immunohistochemistry, Expressing, Knockdown, Over Expression, Injection, shRNA, Standard Deviation

Journal: Journal of Cancer

Article Title: EXOSC5 promotes proliferation of gastric cancer through regulating AKT/STAT3 signaling pathways.

doi: 10.7150/jca.69166

Figure Lengend Snippet: Figure 7. Schematic model of EXOSC5/AKT/STAT3 axis in GC. EXOSC5 promotes growth and survival in GC by regulating cell cycle proteins (P21, P27, Cyclin D1) via AKT and STAT3 pathways.

Article Snippet: The primary antibodies are as following: EXOSC5 (Proteintech, 15627-1-AP); p21 (Proteintech, 10355-1-AP); p27 (Proteintech, 25614-1-AP); cyclin D1 (CST, 2922S); p-AKT (CST, 9271S); AKT (CST, 9272S); p-STAT3 (CST, 9145); AKT (CST, 9272S).

Techniques:

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 5. Effect of SER5 on HIV-1 particles pseudotyped with different envelopes (A) HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with envelopes of HIV-1 BaL (red), FeLV-A (green), FeLV-B (blue) or amphotropic (ampho) Env (orange) were produced in the presence of increasing amounts of feline SERINC5 (feSER5) or (B) human SERINC5 (huSER5) (0, 100, 200 or 400 ng). Viral infectivity was determined after normalization for reverse transcriptase activity by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001 to 0.01 very significant (**), 0.01 to 0.05 significant (*), >0.05 not significant (ns). (C) SERINC5 stability in the presence of increasing amounts of FeLV-B envelope. HEK293T cells were transfected with fe/huSER5 and increasing amounts of FeLV-B envelope expression plasmids or empty vector as control. Cells were harvested and analyzed by immunoblotting using anti-p85/70, anti-HA and anti-tubulin antibodies. a p85/70 detects envelope (SU-TM p85) and SU only (p70), a HA detects HA-SER5.

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Infection, Reverse Transcription, Activity Assay, Luciferase, Transfection, Expressing, Control, Western Blot

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 6. Generation of FeLV glycoGag expression constructs. (A) Sequences of FeLV-A and FeLV-B glycoGags. The highlighted amino acids correspond to disagreements, which are colored according to their side chain chemistry. Cytoplasmic and transmembrane domains are indicated. (B) Plasmids expressing the HA-tagged FeLV glycoGag were produced by amplifying the glycoGag from pFGA-5 and pFGB (FeLV-A and FeLV-B glycoGag respectively), yielding a 9.6 kDa protein. (C) HEK293T cells were transfected with the generated expression plasmids, harvested two days post-transfection and analyzed by immunoblotting using anti-HA and anti-tubulin antibodies.

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Expressing, Construct, Produced, Transfection, Generated, Western Blot

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 7. Effect of FeLV glycoGag on HIV-1 in the presence of SER5. (A) HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with HIV-1 BaL envelope were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) together with FeLV-A glycoGag, FeLV-B glycoGag or empty vector. Particles were normalized by RT activity and used for infections. The infectivity determined by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns). (B) Effect of SERINC and glycoGag on HIV-1 cell-to-cell transmission. HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with HIV-1 envelope BaL were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) and FeLV-A or -B glycoGag. After transfection, virus producer HEK293T cells were co-cultured with HEK293T transfected with expression plasmids for human CD4 and human CCR5. The infectivity determined by quantification of luciferase activity of a vector that is detectable only after reverse transcription of the spliced luciferase gene. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001– 0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Activity Assay, Infection, Luciferase, Transmission Assay, Transfection, Virus, Cell Culture, Expressing, Reverse Transcription

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 8. Effect of FeLV glycoGag or FeLV-B envelope on FIV in the presence of SER5. FIV particles (3- plasmid system) pseudotyped with FIV EE14 envelope were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) and FeLV glycoGag or in the absence of FIV EE14 envelope and glycoGag and pseudotyped by FeLV-B envelope. Particles were normalized by RT activity and used for infection. The infectivity determined by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Activity Assay, Infection, Luciferase

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 9. Comparison of delta Ct values of feSER5 in transfected HEK293T cells and in cat cells and surface expression of feSER5 in the presence of different FeLV proteins. (A) HEK293T cells were transfected with different amounts of feSER5 plasmid. The endogenous feSER5 were evaluated in feline CRFK cell lines and PBMC or macrophages from one cat. (B) HEK293T cells were cotransfected with feSER5-iHA and empty plasmid, Nef, MLV glycoGag , FeLV-A/B glycoGag or FeLV-A/B envelope. The percent of feSER5 positive cells was evaluated by flow cytometry analysis and the total of feSER5 positive cells were scaled to 100%. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01– 0.05 significant (*), >0.05 not significant (ns). (C) Representative plots of one of three experiments showing the surface expression of feSER5-iHA (HA-Alexa Fluor 488). Green fluorescence was quantified using the FITC-A channel vs. FSC-A (10,000 cells were counted).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Comparison, Transfection, Expressing, Plasmid Preparation, Cytometry

Journal: Journal of Cancer

Article Title: Oxymatrine Synergistically Potentiates the Antitumor Effects of Cisplatin in Human Gastric Cancer Cells.

doi: 10.7150/jca.28532

Figure Lengend Snippet: Figure 6. OMT and CDDP act synergistically to inhibit the AKT/ERK pathway. (a) Western blotting assay was used to analysis the expression level of cyclin D1, p21, p27, AKT, p-AKT, ERK and p-ERK. (b) The densitometry analysis of every factor was performed, normalized with the corresponding GAPDH content. *P < 0.01 versus OMT or CDDP alone group.

Article Snippet: AKT, p-AKT, ERK, p-ERK, cyclinD1, p21 and p27 antibodies were purchased from Cell Signaling Technology (MA, USA), and the GAPDH antibody was from Kangchen Bio-tech (Shanghai, China).

Techniques: Western Blot, Expressing

Journal: Chemical & pharmaceutical bulletin

Article Title: Synthesis, Antiproliferative Activity and Molecular Docking Analysis of Both Enantiomerically Pure Decursin Derivatives as Anticancer Agents.

doi: 10.1248/cpb.c23-00718

Figure Lengend Snippet: Figure 3. Induction of cell cycle arrest and apoptosis by decursin or (S)-2d (MRC-D-004) on A549 lung cancer cells: (A) The protein levels of CDK2, cyclin D1, cyclin D3, p27Kip1 and p-cdc2 (Tyr15) were determined by western blotting assays. Data are represented as mean ± SD of three experiments. Decursin and (S)-2d were compared with vehicle control by Student's t-test. ***P<0.001 (both compounds) vs. vehicle control. (B) cell lysates were prepared for western blot analysis against caspase-8, -9, PARP, and GAPDH. Data are represented as mean ± SD of three experiments. Decursin and were compared with vehicle control by Student's t-test. **P<0.01 (decursin) and *P<0.05 [(S)-2d] vs. vehicle control, respectively.

Article Snippet: The blocked membranes were incubated with primary antibody for CXCR7 (PA3-069, 1:5000, Thermo Fisher Scientific, USA), GAPDH (5174, 1:1000, CST, USA), p-JAK1 (3331, 1:1000, CST, USA), JAK1 (3332, 1:1000, CST, USA), p-JAK2 (4406, 1:1000, CST, USA), JAK2 (3230, 1:1000, CST, USA), p-STAT3 (9145, Chemical and Pharmaceutical Bulletin Advance Publication 1:1000, CST, USA), STAT3 (4904, 1:1000, CST, USA), CDK2 (2546, 1:1000, CST, USA), Cyclin D1 (2978, 1:1000, CST, USA), Cyclin D3 (2936, 1:1000, CST, USA), p27 KIP1 (3686, 1:1000, CST, USA), p-cdc2 (4539, 1:1000, CST, USA), Cleaved caspase 8 (9496, 1:1000, CST, USA), Cleaved caspase 9 (7237, 1:1000, CST, USA) and PARP (9542, 1:1000, CST, USA) All primary antibodies were sued with overnight incubation at 4 °C.

Techniques: Western Blot, Control

Journal: Oncogene

Article Title: Induction of S-phase arrest and p21 overexpression by a small molecule 2[[3-(2,3-dichlorophenoxy)propyl] amino]ethanol in correlation with activation of ERK.

doi: 10.1038/sj.onc.1207645

Figure Lengend Snippet: Figure 3 Effect on p21 and p27 expression in DLD-1 cells. (a) Cells were treated with 20-mM of 2,3-DCPE and harvested at various times. Cells treated with DMSO were used as a control for each time point. (b) Cells were treated with different concentration of 2,3-DCPE for 24 h. Cells treated with DMSO for 24 h were used as a control

Article Snippet: Rabbit anti-human p27, ERK, phospho-ERK, p38, phospho-p38, JNK and phospho-JNK, and mouse anti-human p21 were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Control, Concentration Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Tyrosine phosphatase SHP-2 is a regulator of p27(Kip1) tyrosine phosphorylation.

doi: 10.4161/cc.7.24.7260

Figure Lengend Snippet: Figure 1. SHP-2 interacts with p27Kip1 and G-CSF receptor and translocates to the nucleus on G-CSF-stimulation in NB4 cells. (A) Lysates of NB4 cells untreated or stimulated with G-CSF were precipitated with antibody to p27Kip1, the G-CSF receptor or SHP-2 and probed as indicated. In this analysis two major G-CSF receptor isotypes (arrows) were detected. Control immunoprecipitations with an antibody to human IgG excluded the possibility of unspecific binding. (B) p27Kip1 and SHP-2 were expressed in rabbit reticulocyte lysate by in vitro translation, both lysates were mixed together and incubated with antibody to p27Kip1 or SHP-2, and probed as indicated. Control reticulocyte lysate containing in vitro translated p27Kip1 or SHP-2 was loaded. (C) NB4 cells unstimulated or stimulated with G-CSF for 15 and 30 minutes were fractionated, a total of 100 μg lysate from cytosolic fraction C and nuclear fraction N was probed as indicated. Transcription factor PU.1 and Src kinase were used as markers for nuclear and cytosolic fraction respectively. (D) NB4 cells unstimulated or stimulated with G-CSF for 15 and 30 minutes were fractionated. Lysates were precipitated with antibody to SHP-2 or p27Kip1 and probed as indicated.

Article Snippet: For functional silencing experiments NB4 cells were treated with SHP-2 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-36488, Santa Cruz, Heidelberg, Germany), p27Kip1 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-29429) or nonsense siRNA (sc-37007, Santa Cruz) by ‘Magnet assisted Transfection’ (MATra, IBA, Göttingen, Germany) according to manufacturers instructions.

Techniques: Control, Binding Assay, In Vitro, Incubation

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Tyrosine phosphatase SHP-2 is a regulator of p27(Kip1) tyrosine phosphorylation.

doi: 10.4161/cc.7.24.7260

Figure Lengend Snippet: Figure 2. SHP-2 abrogates nuclear translocation of tyrosine-phosphorylated p27Kip1 in transiently transfected NIH/3T3 fibroblasts. NIH/3T3 cells were transiently transfected with plasmids containing p27Kip1, SHP-2 (wild-type as well as catalytically inactive CS point mutation) or constitutive active c-Abl (Abl-PP) sequence. After fixing, the cells were probed with antibodies against p27Kip1 and SHP-2 and visualized with rhodamine-conju- gated anti-mouse IgG or Alexa Flour 488-conjugated anti-rabbit IgG by fluorescent microscopy, respectively.

Article Snippet: For functional silencing experiments NB4 cells were treated with SHP-2 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-36488, Santa Cruz, Heidelberg, Germany), p27Kip1 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-29429) or nonsense siRNA (sc-37007, Santa Cruz) by ‘Magnet assisted Transfection’ (MATra, IBA, Göttingen, Germany) according to manufacturers instructions.

Techniques: Translocation Assay, Transfection, Mutagenesis, Sequencing, Microscopy

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Tyrosine phosphatase SHP-2 is a regulator of p27(Kip1) tyrosine phosphorylation.

doi: 10.4161/cc.7.24.7260

Figure Lengend Snippet: Figure 3. SHP-2 modulates tyrosine dephosphorylation of p27Kip1 in NIH/3T3 fibroblasts; expression of catalyti- cally inactive SHP-2 and SHP-2 knockdown prevent G-CSF- mediated tyrosine dephosphorylation of p27Kip1 in NB4 cells. (A) NIH/3T3 cells were transiently transfected with plasmids containing p27Kip1, SHP-2 (wild-type as well as catalytically inactive CS point mutation) or constitutive active c-Abl (Abl- PP) sequence. p27Kip1 immunoprecipitations were performed and probed with antibodies as indicated. As control for actin, p27Kip1, Abl-PP and SHP-2 expression a total of 50 μg of lysate was also detected. (B) NB4 cells were transfected with expression plasmid containing the sequence of catalytically inactive SHP-2 (SHP-2 CS) or with a non-coding vector (Mock) and stimulated with G-CSF for 30 minutes after 48 hours. p27Kip1 immunoprecipitations were performed and probed with antibodies as indicated. Additionally, the expression of SHP-2 CS was examined by probing a total of 50 μg of lysate as indicated. (C) NB4 cells were transfected with SHP-2 siRNA or nonsense siRNA (siRNA Ctrl.) and stimulated with G-CSF for 30 minutes after 48 hours. p27Kip1 immunoprecipi- tations were performed and probed with antibodies as indi- cated. Additionally, the effect of SHP-2 siRNA was examined by probing a total of 50 μg of lysate as indicated.

Article Snippet: For functional silencing experiments NB4 cells were treated with SHP-2 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-36488, Santa Cruz, Heidelberg, Germany), p27Kip1 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-29429) or nonsense siRNA (sc-37007, Santa Cruz) by ‘Magnet assisted Transfection’ (MATra, IBA, Göttingen, Germany) according to manufacturers instructions.

Techniques: De-Phosphorylation Assay, Expressing, Knockdown, Transfection, Mutagenesis, Sequencing, Control, Plasmid Preparation

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Tyrosine phosphatase SHP-2 is a regulator of p27(Kip1) tyrosine phosphorylation.

doi: 10.4161/cc.7.24.7260

Figure Lengend Snippet: Figure 4. SHP-2 modulates p27Kip1 binding with cdks; PTP activity of SHP-1 and SHP-2 is mediated by G-CSF in NB4 cells. (A) NIH/3T3 cells were transiently transfected with plasmids containing the sequence of p27Kip1, c-Abl (Abl-PP) or wild-type SHP-2, and lysate from transfected cells was immu- noprecipitated and probed as indicated. (B–D) For PTP activity assays NB4 cells were stimulated with G-CSF, harvested at time points indicated, lysed and SHP-2, SHP-1, p27Kip1 and G-CSF receptor were immunoprecipitated. The assays were initiated by addition of the artificial substrate para-nitrophenyl phosphate (pNPP), and PTP activity was detected by measuring the conversion of pNPP at 415 nm. The PTP activity was corrected for the amount of immunoprecipitated protein present in the assay. Error bars indicate SEM (n = 5).

Article Snippet: For functional silencing experiments NB4 cells were treated with SHP-2 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-36488, Santa Cruz, Heidelberg, Germany), p27Kip1 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-29429) or nonsense siRNA (sc-37007, Santa Cruz) by ‘Magnet assisted Transfection’ (MATra, IBA, Göttingen, Germany) according to manufacturers instructions.

Techniques: Binding Assay, Activity Assay, Transfection, Sequencing, Immunoprecipitation

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Tyrosine phosphatase SHP-2 is a regulator of p27(Kip1) tyrosine phosphorylation.

doi: 10.4161/cc.7.24.7260

Figure Lengend Snippet: Figure 5. SHP-2 but not SHP-1 induces tyrosine dephosphorylation of endogenous p27Kip1. (A–C) Dephosphorylation assays were performed with GST-SHP-1 and GST- SHP-2 fusion protein. Therefore, NB4 cells were lysed and immunoprecipited with antibodies to p27Kip1, JAK2 and cdk2. The pre- cipitates were incubated in dephosphorylation assay buffer alone (w/o) or supplemented with 1 μg GST-SHP-1 or GST-SHP-2 fusion protein for 1 or 5 minutes at 30°C. Western blots were detected as indicated.

Article Snippet: For functional silencing experiments NB4 cells were treated with SHP-2 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-36488, Santa Cruz, Heidelberg, Germany), p27Kip1 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-29429) or nonsense siRNA (sc-37007, Santa Cruz) by ‘Magnet assisted Transfection’ (MATra, IBA, Göttingen, Germany) according to manufacturers instructions.

Techniques: De-Phosphorylation Assay, Incubation, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Tyrosine phosphatase SHP-2 is a regulator of p27(Kip1) tyrosine phosphorylation.

doi: 10.4161/cc.7.24.7260

Figure Lengend Snippet: Figure 6. SHP-2 modulates stability of p27Kip1. Cycloheximide (CHX) chase experiment of p27Kip1 with unstimulated or G-CSF-stimulated NB4 cells and NB4 cells transiently transfected with wild-type SHP-2 or catalytically inactive SHP-2 CS, and cells transfected either with nonsense siRNA or with SHP-2-specific siRNA.

Article Snippet: For functional silencing experiments NB4 cells were treated with SHP-2 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-36488, Santa Cruz, Heidelberg, Germany), p27Kip1 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-29429) or nonsense siRNA (sc-37007, Santa Cruz) by ‘Magnet assisted Transfection’ (MATra, IBA, Göttingen, Germany) according to manufacturers instructions.

Techniques: Transfection

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Tyrosine phosphatase SHP-2 is a regulator of p27(Kip1) tyrosine phosphorylation.

doi: 10.4161/cc.7.24.7260

Figure Lengend Snippet: Figure 7. SHP-2 modulates G1 to S phase progression in NB4 cells. (A) NB4 cells were transfected with siRNA as indicated, stained with propidium iodide (PI) after 48 hours, the DNA content was determined by flow cytometry, and the percentages of cells in each phase of the cell cycle were calculated. The shown diagrams and the calculated results are representative of three indepen- dent experiments. (B) The effect of p27Kip1 siRNA was examined by probing a total of 50 μg of lysate as indicated.

Article Snippet: For functional silencing experiments NB4 cells were treated with SHP-2 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-36488, Santa Cruz, Heidelberg, Germany), p27Kip1 siRNA (pool of 3 target-specific 20–25 nt siRNAs, sc-29429) or nonsense siRNA (sc-37007, Santa Cruz) by ‘Magnet assisted Transfection’ (MATra, IBA, Göttingen, Germany) according to manufacturers instructions.

Techniques: Transfection, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Expression and roles of p16 INK4a , p21 Cip1 and p27 Kip1 in the response of lung adenocarcinoma cell line models to long-term treatment with Dex. H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells were treated with Dex (100 nM) for the indicated number of days and whole cell lysates were probed by western blot for p16 INK4a , p21 Cip1 and p27 Kip1 with GAPDH as the loading control (Panel A). H1299GR Clone 4 cells were transduced with either non-targeted control shRNA or P21 Cip1 shRNA and treated with Dex (100 nM) for the indicated number of days, when the cells were harvested and whole cell lysates were extracted and probed by western blot for p21 Cip1 , p27 Kip1 and GAPDH (loading control) (Panel B). In parallel, cells treated as described for Panel B, were plated for colony formation in 6-well plates in triplicates without further treatment (Panel C). H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or p27 Kip1 siRNA and treated with Dex (100 nM) for the indicated number of days, when the cells were harvested and whole cell lysates were probed by western blot for p27 Kip1 or GAPDH (loading control) (Panel D). In parallel, cells transfected as described for Panel D, and treated with Dex (100 nM) were plated on days 0 and 5 to measure colony formation in 6-well plates in triplicates without further treatment (Panel E) and also harvested on days 0 and 3 of treatment for measuring cell size (Panel F). H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or p27 Kip1 siRNA and 24 h later they were treated with Dex (100 nM) or vehicle for 3 days and the cells were stained to assess expression of β- galactosidase (blue staining) (Panel G, left); in parallel, the treated cells were lysed and analyzed by western blot for p27 expression (Panel G, right). H1299GR Clone 4 cells were transduced with either non-targeted control vector or pLenti-p27 expression vector and after the indicated number of days, whole cell lysates were probed by western blot for p27 Kip1 or GAPDH (loading control) (Panel H). In parallel, cells were harvested for measuring the cell size (Panel I) and also plated to measure colony formation in 6-well plates in triplicate (Panel J). Panel C: *P, 0.0004; **P, 0.0006; § P, 0.0008; ‡ P, 0.00015. Panel E: *P, 0.016. Panel J: *P, 0.015; § P, 0.00006; ‡ P, 0.00007.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

Techniques: Expressing, Western Blot, Control, Transduction, shRNA, Transfection, Staining, Plasmid Preparation

Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Mechanism of accumulation of p27 Kip1 in response to long-term Dex treatment in high GR expressing cells. H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or a combination of FOXO3 and FOXO4 siRNAs and treated with Dex (100 nM) for the indicated number of days, when whole cell lysates were probed by western blot for FOXO3, FOXO4, p27 Kip1 and GAPDH (loading control) (Panel A). H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells were treated with Dex (100 nM) for the indicated number of days, when RNA was extracted and relative expression levels of p27 Kip1 mRNA were measured using real time RT-PCR (Panel B). H1299GR Clone 4 cells were pre-treated with vehicle or Dex (100 nM) for 72 h. The cells were then treated with 1ug/ml of actinomycin D and at the time points indicated, RNA was harvested from the cells and p27 Kip1 mRNA was measured by real time RT-PCR to determine p27 Kip1 mRNA turnover rates. The mRNA values are plotted relative to the values at the time of addition of actinomycin D (Panel C). Panel B (H292): *P, 0.01; § P, 0.008. Panel B (A549): *P, 0.02; § P, 0.0001. Panel B (H1299GRα clone2): *P, 0.0001; ‡ P, 0.0007, § P, 0.001. Panel B (H1299GRα clone4): *P, 0.0001; ‡ P, 0.001; § P, 0.0001.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

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

Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Effect of Dex on mouse tumor xenografts expressing low or high levels of GR. SCID mice bearing xenografts of parental H1299 cells (Panel A) or H1299GR Clone 4 cells (Panel B) were implanted with either placebo or Dex (2.5 mg) slow-release pellets at the time indicated by the arrow. The tumor growth was monitored via caliper measurements and tumor volumes were calculated using the formula noted in the Methods section. At the time of sacrifice, residual H1299GR Clone 4 tumors were harvested from the placebo (Panel C, left) and Dex (Panel C, right) treated mice and stained to ensure GR expression and to quantify p27 Kip1 by immunohistochemistry (Panel D). The scoring criteria based on staining intensity and percent positivity, as outlined in the Methods section. The parental H1299 cell tumor xenografts harvested at the end of placebo or Dex treatments in Panel A were also examined by immunohistochemistry for GR and p27 Kip1 expression (Panel E). Panel B: *P, 0.08; § P, 0.016; ‡ P, 0.0015.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification

doi: 10.1038/s41467-017-01742-7

Figure Lengend Snippet: GJA4 regulates endothelial cell cycle arrest via CDKN1B. a CDKN1B mRNA expression was significantly reduced in Gja4 −/− P6 retinal endothelial cells (CD31+/CD45−) (mean relative mRNA expression ± SEM vs. Gja4 +/− littermate controls; n = 3 ( Cdk4 , Gja4 −/− ; Cdk6 , Gja4 −/− Ccne1 , Gja4 +/− ; Ccne2 , Gja4 +/− ), n = 4 ( Cdk4 , Gja4 +/− ; Cdk6 , Gja4 +/− ; Ccnb1 ; Cdkn1a , Gja4 −/− ), n = 5 ( Ccne1 , Gja4 −/− ), n = 6 ( Ccne2 , Gja4 −/− ; Cdkn1a , Gja4 +/− ; Ccnd1 , Gja4 −/− ), n = 8 ( Cdk2 ; Tp53 , Gja4 −/− ), n = 9 ( Ccnd1 , Gja4 +/− ; Cdkn1b ; Tp53 , Gja4 −/− ); individual values plotted where n < 5). b CDKN1B expression was detected in Gja4 +/+ , but not Gja4 −/− , endothelial cells of remodeling retinal vessels. (Colors: PECAM1 (red), CDKN1B (green); scale bar: 50 µm). c CDKN1B protein was elevated in HUVEC by 16 h FSS, and this effect was abolished by si- GJA4 (mean densitometry ± SEM vs. Static; n = 3 for all groups; one-way ANOVA: p = 0.002, asterisks indicate p < 0.05 in post hoc t -test). Uncropped western blots presented in Supplementary Fig. . d Constitutive GJA4 expression (lenti- Gja4 ) arrested endothelial cells in G1 and reduced actively cycling cells in S/G2/M, while si- CDKN1B had the opposite effect, regardless of GJA4 expression (mean difference in cell cycle % ± SEM vs. Control; n = 6; one-way ANOVA: p = 0.0002 (G1), p = 0.0003 (S/G2/M), asterisks indicate p < 0.05 in post hoc t -test vs. si-Ctrl + lenti-Ctrl). e GJA4 knockdown (si- GJA4 ) reduced the proportion of endothelial cells in G1 and increased the proportion in S/G2/M, and constitutive expression of CDKN1B (lenti- CDKN1B ) had the opposite effect, regardless of GJA4 expression (mean difference in cell cycle % ± SEM vs. Control; n = 3 for all groups; one-way ANOVA: p = 0.002 (G0), p < 0.0001 (G1), p < 0.0001 (S/G2/M), asterisks indicate p < 0.05 in post hoc t -tests vs. si-Ctrl+ lenti-Ctrl). f CDKN1B phosphorylation at serine 10 (pCDKN1B (S10)) and total protein levels were reduced by si- GJA4 and rescued with lenti- Gja4 . Uncropped blots presented in Supplementary Fig. . g MAPK/ERK signaling inhibition by 1 h treatment with 20 µM U0126, an inhibitor of MEK1/2, blocked GJA4-dependent CDKN1B phosphorylation at serine 10 and reduced total CDKN1B protein levels. Uncropped blots presented in Supplementary Fig.

Article Snippet: For siRNA knockdown, cells pre-conditioned with EGM-2 (Lonza) for >16 h were transfected with 20 nM si- GJA4 (Dharmacon L-011669-00), si- CDKN1B (Dharmacon L-040178-00), si- NOTCH1 (Dharmacon L-007771-00), si- NOTCH4 (Dharmacon L-011883-00) or control siRNA (Ambion AM4611) for >96 h. For lentiviral transduction, HUVEC were pre-infected for 48 h with lentivirus containing mouse Gja4 (lenti- Gja4 ) , human CDKN1B (lenti- CDKN1B , cloned from Addgene 14049) or empty vector in presence of 10 μg/mL polybrene (Sigma).

Techniques: Expressing, Western Blot, Control, Knockdown, Phospho-proteomics, Inhibition

Journal: Nature Communications

Article Title: Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification

doi: 10.1038/s41467-017-01742-7

Figure Lengend Snippet: Endothelial cell cycle arrest, per se, enables arterial gene expression. a Treatment of HUVEC with 10 µM clotrimazole or 2 µM palbociclib reduced RB1, phosphorylated RB1 (pRb1), and E2F1, suggestive of G1 arrest. Clotrimazole tended to upregulate CDKN1B expression and preserve GJA4 expression, and CDK4 expression was lost only with CDK4/6i treatment. Uncropped blots presented in Supplementary Fig. . b Using FACS to assess cell cycle distribution, clotrimazole was found to arrest HUVEC in G1, even when CDKN1B was knocked down (via si- CDKN1B ) (mean difference in cell cycle % ± SEM vs. DMSO; n = 3 (Clotrimazole), n = 5 (DMSO), n = 6 (Clotrimazole+ si- CDKN1B ); individual values plotted where n < 5; one-way ANOVA: p = 0.007 (G1), p = 0.03 (S/G2/M); asterisks indicate p < 0.05 in post hoc t -tests). c Clotrimazole upregulated EFNB2 and GJA5 regardless of CDKN1B expression (mean relative mRNA expression ± SEM vs. DMSO; n = 4 (Clotrimazole, p27), n = 5 (Clotrimazole, EFNB2 ; Clotrimazole, GJA5 ), n = 7 (Clotrimazole+ si-CDKN1B), n = 8 (DMSO); individual values plotted where n < 5; one-way ANOVA: p = 0.03 ( EFNB2 , GJA5 ), p = 0.0007 ( CDKN1B )). d Palbociclib treatment also arrested endothelial cells in G1 (mean difference in cell cycle % ± SEM vs. DMSO; n = 3 for all groups; Students’ t -test: p = 0.002 (G1), p < 0.0001 (S/G2/M)), e upregulated EFNB2 and GJA5 (Cx40) mRNA levels, abolished CDK4 expression, and had no effect on GJA4 (Cx37) and CDKN1B (p27), (mean relative mRNA expression ± SEM vs DMSO; n = 9 ( GJA4 ), n = 12 ( EFNB2 ), n = 14 ( GJA5 ), n = 16 ( CDKN1B ; CDK4 ); Students’ t -test: p = 0.02 ( EFNB2 , GJA5 ), p < 0.0001 ( CDK4 )). f Endothelial cells within arteries and surrounding plexi of P6 retinas from Cdt1 -mOrange+ reporter mice were predominantly in G1 phase, whereas endothelial cells in adjacent veins were not. (Colors: IB4 (red), ERG (blue), pH3 (magenta), CDT1 (green); scale bar: 100 µm). g We hypothesize that in remodeling vessels, arterial shear activates a novel Notch-Cx37-p27 signaling pathway that promotes endothelial cell cycle arrest to enable arterial gene expression

Article Snippet: For siRNA knockdown, cells pre-conditioned with EGM-2 (Lonza) for >16 h were transfected with 20 nM si- GJA4 (Dharmacon L-011669-00), si- CDKN1B (Dharmacon L-040178-00), si- NOTCH1 (Dharmacon L-007771-00), si- NOTCH4 (Dharmacon L-011883-00) or control siRNA (Ambion AM4611) for >96 h. For lentiviral transduction, HUVEC were pre-infected for 48 h with lentivirus containing mouse Gja4 (lenti- Gja4 ) , human CDKN1B (lenti- CDKN1B , cloned from Addgene 14049) or empty vector in presence of 10 μg/mL polybrene (Sigma).

Techniques: Gene Expression, Expressing, Shear

Journal: Nature Communications

Article Title: Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification

doi: 10.1038/s41467-017-01742-7

Figure Lengend Snippet: FSS-NOTCH-GJA4-CDKN1B axis regulates arterial identity genes. a Disorganized or absent SMA investment was observed in Gja4 −/− and WT + DAPT mice, compared to WT controls. (Colors: PECAM1 (red), SMA (white); scale bar: 100 µm; symbols: a = artery, v = vein, R = remodeling, M = mature). b Radial SMA investment of arteries, as well as c radial distance of GJA5 (Cx40) expression, were reduced in Gja4 −/− and DAPT-treated animals (mean radial distance as % of total vascular outgrowth ± SEM vs WT; n = 3 (WT + DAPT; WT, GJA5), n = 4 (WT, SMA), n = 6 ( Gja4 −/− ); one-way ANOVA: p = 0.03 (SMA), p = 0.04 (GJA5); asterisks indicate p < 0.05 in post hoc t -tests vs. WT). d DLL4 activation of NOTCH signaling upregulated EFNB2 (EphrinB2) and GJA5 , and this effect was abolished with si- GJA4 or si- CDKN1B (mean relative mRNA expression ± SEM vs. PBS; n = 3 (si- GJA4 ; si- CDKN1B, EFNB2 ), n = 4 (si-Ctrl, GJA5 ; si- CDKN1B , GJA5 ), n = 4 (si-Ctrl, EFNB2 ); Students’ t -test: p = 0.02 (si-Ctrl, EFNB2 ), p = 0.04 (si-Ctrl, GJA5 ), p = 0.0005 (si- GJA4 , GJA5 ), p < 0.0001 (si- CDKN1B , GJA5 )). e NOTCH activation was maximal with 1 h exposure to 18 dynes/cm 2 shear (mean NICD R.F.U. ± SEM; n = 7000 cells, representative of N = 3). f Expression of GJA4 (Cx37), GJA5 (Cx40) and EFNB2 (EphrinB2) were also maximal at 18 dynes/cm 2 (mean relative mRNA expression ± SE; n = 3, representative of 4 experiments). g Knockdown of NOTCH1, GJA4 or CDKN1B reduced basal and 10 h FSS-induced upregulation of EFNB2 and GJA5 (mean relative mRNA expression ± SEM vs. Static; n = 3 technical replicates; representative of two biological replicates)

Article Snippet: For siRNA knockdown, cells pre-conditioned with EGM-2 (Lonza) for >16 h were transfected with 20 nM si- GJA4 (Dharmacon L-011669-00), si- CDKN1B (Dharmacon L-040178-00), si- NOTCH1 (Dharmacon L-007771-00), si- NOTCH4 (Dharmacon L-011883-00) or control siRNA (Ambion AM4611) for >96 h. For lentiviral transduction, HUVEC were pre-infected for 48 h with lentivirus containing mouse Gja4 (lenti- Gja4 ) , human CDKN1B (lenti- CDKN1B , cloned from Addgene 14049) or empty vector in presence of 10 μg/mL polybrene (Sigma).

Techniques: Expressing, Activation Assay, Shear, Knockdown