Journal: Scientific Reports

Article Title: Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

doi: 10.1038/srep39700

Figure Lengend Snippet: Western blot analysis of nuclear and cytoplasmic fractions of PEDV-infected Vero E6 cells with an anti-GAPDH mAb ( A ), anti-PCNA mAb ( B ), and anti-N mAb ( C ). ( A,B and C ) Nuc, nuclear fraction of PEDV-infected cells; Cyt, cytoplasmic fraction of PEDV-infected cells. ( C ) Moc, mock-infected cells; Inf, PEDV-infected cells. The arrowheads indicate purified bands that are the same sizes as GAPDH ( A ), PCNA ( B ), and N ( C ) proteins. ( D ) Western blot analysis of N protein in nuclear and cytoplasmic fractions of PEDV-infected Vero E6 cells at different times with an anti-GAPDH mAb, anti-PCNA mAb, and anti-N mAb. Nuc, nuclear fraction of PEDV-infected cells; Cyt, cytoplasmic fraction of PEDV-infected cells.

Article Snippet: The membrane was soaked in blocking buffer (PBS containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: mouse anti-GAPDH mAb (1:10,000) (G8795; Sigma), mouse anti-PCNA mAb (1:200) (BM0104; BOSTER), mouse anti-N mAb (1:500), mouse anti-GST mAb (1:1000) (AG768; Beyotime), mouse anti-actin mAb (1:5000) (A5441; Sigma), mouse anti-GFP mAb (1:10,000) (66002–1–1 g; Proteintech), rabbit anti-Flag mAb (1:2000) (20543–1-AP; Proteintech), or mouse anti Myc mAb (1:2000) (66004–1–1 g; Proteintech), rabbit anti-caspase-3 mAb (1:1000) (AC030; Beyotime).

Techniques: Western Blot, Infection, Purification

Journal: Scientific Reports

Article Title: Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

doi: 10.1038/srep39700

Figure Lengend Snippet: ( A ) The NPM1 fusion protein as a marker of the nucleolus is colored red. The nucleolar localization of N in transfected cells was clearly observed at 42–42.5 hpt. As shown, a small amount of N was observed in the nucleolus at 42 hpt (t = 30 min). N protein accumulated continuously in the nucleolus of transfected cells until t = 60 min and was exported from the nucleolus at t = 61–65 min. This figure shows snapshots of the cells from the time-lapse movie ( in the ). Data are representative of one of three independent experiments. Real-time visualization of the kinetics of the nucleolar localization of N protein indicated that the process was rapid, taking only 30 min in total. ( B ) Knockdown of NPM1 protein levels following siRNA treatment. Vero E6 cells transfected with no siRNA (Mock), scrambled siRNA (siScr), left untreated (No treat) or with different concentrations (mM) of siRNAs targeting NPM1 (siNPM1) (as indicated at the top of each lane) were harvested 48 hpt. Endogenous NPM1 protein levels were detected by immunoblotting using antibodies directed against the indicated proteins. ( C and D ) Western blot analysis of Myc-N protein in nuclear and cytoplasmic fractions of NPM1-knockdown cells ( C ) or Ectopic NPM1-overexpression cells ( D ) at 48 hpt with anti-GAPDH mAb, anti-PCNA mAb, anti-NPM1 mAb, anti-Myc mAb and anti-Flag mAb. Nuc, nuclear fraction; Cyt, cytoplasmic fraction; cell, whole cells. Densitometric data for Nuc/Cell (Myc-N) from three independent experiments are expressed as mean ± SD.

Article Snippet: The membrane was soaked in blocking buffer (PBS containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: mouse anti-GAPDH mAb (1:10,000) (G8795; Sigma), mouse anti-PCNA mAb (1:200) (BM0104; BOSTER), mouse anti-N mAb (1:500), mouse anti-GST mAb (1:1000) (AG768; Beyotime), mouse anti-actin mAb (1:5000) (A5441; Sigma), mouse anti-GFP mAb (1:10,000) (66002–1–1 g; Proteintech), rabbit anti-Flag mAb (1:2000) (20543–1-AP; Proteintech), or mouse anti Myc mAb (1:2000) (66004–1–1 g; Proteintech), rabbit anti-caspase-3 mAb (1:1000) (AC030; Beyotime).

Techniques: Marker, Transfection, Knockdown, Western Blot, Over Expression

Journal: Scientific Reports

Article Title: Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

doi: 10.1038/srep39700

Figure Lengend Snippet: ( A and B ) N protein binding prevents NPM1 proteolytic cleavage. Vero E6 cells were transfected with pMyc-N or empty vector for 24 h and then treated with or without 100 μM of Ac-DEVD-CHO (caspase-3 inhibitor) for 6 h. The cells were treated with or without 250 nm of STS for 18 h. The western blots were probed for freshly extracted proteins with antibodies against NPM1, Myc, GAPDH and caspase-3. Verification of Myc-N induction and equal sample loading are shown by anti-Myc and anti-GAPDH mAbs. CF, cleavage fragment. ( C ) N protein enhances the antiapoptotic effect of NPM1. Vero E6 cells were transfected with pMyc-N or empty vector for 30 h and then treated with or without 250 nm of STS for 18 h. Genomic DNA was loaded on to a 2% agarose gel. Verification of Myc-N induction and equal sample loading are shown by anti-Myc and anti-GAPDH mAbs. ( D ) Vero E6 cells were transfected with pMyc-N or empty vector for 30 h and then treated with or without 250 nm of STS for 18 h, then TUNEL and DAPI staining to examine the apoptotic cell death. Statistical results represent means ± SD of apoptotic cell counts from six different fields (right).

Article Snippet: The membrane was soaked in blocking buffer (PBS containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: mouse anti-GAPDH mAb (1:10,000) (G8795; Sigma), mouse anti-PCNA mAb (1:200) (BM0104; BOSTER), mouse anti-N mAb (1:500), mouse anti-GST mAb (1:1000) (AG768; Beyotime), mouse anti-actin mAb (1:5000) (A5441; Sigma), mouse anti-GFP mAb (1:10,000) (66002–1–1 g; Proteintech), rabbit anti-Flag mAb (1:2000) (20543–1-AP; Proteintech), or mouse anti Myc mAb (1:2000) (66004–1–1 g; Proteintech), rabbit anti-caspase-3 mAb (1:1000) (AC030; Beyotime).

Techniques: Protein Binding, Transfection, Plasmid Preparation, Western Blot, Agarose Gel Electrophoresis, TUNEL Assay, Staining

Journal: Pharmaceutics

Article Title: Antioxidant and Antiproliferative Activity of Finasteride against Glioblastoma Cells

doi: 10.3390/pharmaceutics13091410

Figure Lengend Snippet: Glioblastoma stem-like cells are sensitive to high-dose finasteride. ( A ) Cell morphology of U373 (upper) and T98G (lower) glioblastoma cells under sphere-forming culture condition upon treatment of DMSO or temozolomide (TMZ) or 100 μM finasteride (FIN) for 24 h. ( B ) The mRNA expression of Sox2 in U373 (left) and T98G (right) cells upon vehicle or drug treatment, as measured by quantitative RT-PCR analysis. The mRNA level of vehicle-treated cells was set to 1. ( C ) Immunoblot analysis of β-catenin in U373 (left) or T98G (right) cells after treatment with vehicle or FIN at the indicated concentration for 24 h. GAPDH was used as a loading control. The relative band intensities of β-catenin are shown below. The intensities of vehicle-treated samples were arbitrarily set to 1. ( D ) β-catenin activity was measured in U373 (left) or T98G (right) cells by TOPFlash reporter assay upon treatment of DMSO or 100 μM FIN for 24 h. ( E ) Immunoblot analysis of P62 and LC3 in U373 (left) or T98G (right) cells after vehicle or drug treatment for 24 h. The relative band intensities of P62 and LC3-II are represented. The intensities of vehicle-treated samples were arbitrarily set to 1. * p < 0.05, ** p < 0.01.

Article Snippet: Immunoblot assays were performed with antibodies against GAPDH (Cusabio, Houston, TX, USA, cat no. CSB-MA000184), β-catenin (Thermo Fisher Scientific, Waltham, MA, USA, cat no. MA1-300), phospho H3 at Ser 10 (Cell Signaling, Leiden, The Netherlands, cat no. 3377), phospho AKT (Thermo Fisher Scientific, Waltham, MA, USA, cat no. 700392), total AKT (Cell Signaling, Leiden, The Netherlands, cat no. 9272), SESN2 (Abcam, Cambridge, UK, cat no. ab178518), SOD2 (Cusabio, Houston, TX, USA, cat no. CSB-PA022398LA01HU), PRDX5 (Bethyl Laboratories, Montgomery, TX, USA, cat no. A305-339A), γH2AX (Novus Biologicals, Centennial, CO, USA, cat no. NB100-384), EGFR (Bioworld Technology, Bloomington, MN, USA, cat no. BS1533), P62 (Abcam, Cambridge, UK, cat no. ab56416), phospho P70S6K (Cell Signaling, Leiden, The Netherlands, cat no. 9205), total P70S6K (Cell Signaling, Leiden, The Netherlands, cat no. 9202), phospho 4EBP1 (Cell Signaling, Leiden, The Netherlands, cat no. 2855), and total 4EBP1 (Cell Signaling, Leiden, The Netherlands, cat no. 9644).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Concentration Assay, Control, Activity Assay, Reporter Assay

Journal: Pharmaceutics

Article Title: Antioxidant and Antiproliferative Activity of Finasteride against Glioblastoma Cells

doi: 10.3390/pharmaceutics13091410

Figure Lengend Snippet: High-dose finasteride suppresses the proliferating capacity of glioblastoma cells. ( A ) U373 (upper) and T98G (lower) cells were treated with vehicle or 100 μM finasteride (FIN) for 24 h, and they were immunostained with a phospho histone 3 (Ser 10)-specific antibody (red). Nuclear DAPI (4′, 6-diamidino-2-phenylindole) signal is presented in blue (left). The quantification of phospho histone 3-positive cell proportions in vehicle- or FIN-treated cells (right). ( B ) Immunoblot analysis of phospho histone 3 in U373 (left) or T98G (right) cells after treatment with vehicle or FIN at the indicated concentration for 24 h. GAPDH was used as a loading control. The relative band intensities of phospho histone 3 are shown below. The intensities of vehicle-treated samples were arbitrarily set to 1. ( C ) Immunoblot analysis of phospho histone 3 in U373 (left) or T98G (right) cells upon treatment of DMSO or temozolomide (TMZ) or 100 μM FIN for 24 h. GAPDH was used as a loading control. The relative band intensities of phospho histone 3 are represented. The intensities of vehicle-treated samples were arbitrarily set to 1. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Immunoblot assays were performed with antibodies against GAPDH (Cusabio, Houston, TX, USA, cat no. CSB-MA000184), β-catenin (Thermo Fisher Scientific, Waltham, MA, USA, cat no. MA1-300), phospho H3 at Ser 10 (Cell Signaling, Leiden, The Netherlands, cat no. 3377), phospho AKT (Thermo Fisher Scientific, Waltham, MA, USA, cat no. 700392), total AKT (Cell Signaling, Leiden, The Netherlands, cat no. 9272), SESN2 (Abcam, Cambridge, UK, cat no. ab178518), SOD2 (Cusabio, Houston, TX, USA, cat no. CSB-PA022398LA01HU), PRDX5 (Bethyl Laboratories, Montgomery, TX, USA, cat no. A305-339A), γH2AX (Novus Biologicals, Centennial, CO, USA, cat no. NB100-384), EGFR (Bioworld Technology, Bloomington, MN, USA, cat no. BS1533), P62 (Abcam, Cambridge, UK, cat no. ab56416), phospho P70S6K (Cell Signaling, Leiden, The Netherlands, cat no. 9205), total P70S6K (Cell Signaling, Leiden, The Netherlands, cat no. 9202), phospho 4EBP1 (Cell Signaling, Leiden, The Netherlands, cat no. 2855), and total 4EBP1 (Cell Signaling, Leiden, The Netherlands, cat no. 9644).

Techniques: Western Blot, Concentration Assay, Control

Journal: Pharmaceutics

Article Title: Antioxidant and Antiproliferative Activity of Finasteride against Glioblastoma Cells

doi: 10.3390/pharmaceutics13091410

Figure Lengend Snippet: High-dose finasteride shows the antioxidant potential in glioblastoma cells. ( A ) U373 (upper) and T98G cells were subjected to CellROX staining after treatment with vehicle or finasteride (FIN) at the indicated concentration for 24 h. Nuclear DAPI signal is shown in blue. The relative CellROX signal intensities are shown (right). Each dot means an individual cell. ( B ) Immunoblot analysis of SESN2, SOD2, and PRDX5 in U373 (left) or T98G (right) cells after treatment with vehicle or FIN at the indicated concentration for 24 h. GAPDH was used as a loading control. The relative band intensities of SESN2, SOD2, and PRDX5 are shown below. The intensities of vehicle-treated samples were arbitrarily set to 1. ( C ) U373 (upper) and T98G cells were subjected to TMRE staining after treatment with vehicle or finasteride (FIN) at the indicated concentration for 24 h. Nuclear DAPI signal is shown in blue. The relative TMRE signal intensities are shown (right). Each dot means an individual cell. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Immunoblot assays were performed with antibodies against GAPDH (Cusabio, Houston, TX, USA, cat no. CSB-MA000184), β-catenin (Thermo Fisher Scientific, Waltham, MA, USA, cat no. MA1-300), phospho H3 at Ser 10 (Cell Signaling, Leiden, The Netherlands, cat no. 3377), phospho AKT (Thermo Fisher Scientific, Waltham, MA, USA, cat no. 700392), total AKT (Cell Signaling, Leiden, The Netherlands, cat no. 9272), SESN2 (Abcam, Cambridge, UK, cat no. ab178518), SOD2 (Cusabio, Houston, TX, USA, cat no. CSB-PA022398LA01HU), PRDX5 (Bethyl Laboratories, Montgomery, TX, USA, cat no. A305-339A), γH2AX (Novus Biologicals, Centennial, CO, USA, cat no. NB100-384), EGFR (Bioworld Technology, Bloomington, MN, USA, cat no. BS1533), P62 (Abcam, Cambridge, UK, cat no. ab56416), phospho P70S6K (Cell Signaling, Leiden, The Netherlands, cat no. 9205), total P70S6K (Cell Signaling, Leiden, The Netherlands, cat no. 9202), phospho 4EBP1 (Cell Signaling, Leiden, The Netherlands, cat no. 2855), and total 4EBP1 (Cell Signaling, Leiden, The Netherlands, cat no. 9644).

Techniques: Staining, Concentration Assay, Western Blot, Control

Journal: Pharmaceutics

Article Title: Antioxidant and Antiproliferative Activity of Finasteride against Glioblastoma Cells

doi: 10.3390/pharmaceutics13091410

Figure Lengend Snippet: Finasteride does not alter EGFR expression. ( A ) Relative amount of luminance from U373 (left) and T98G (right) cells transfected with Gaussia luciferase (GLuc) reporter plasmid-containing EGFR promoter upon treatment of DMSO or finasteride (FIN) at the indicated concentration for 24 h. n.s. , not significant. ( B ) The mRNA expression of Egfr in U373 (left) and T98G (right) cells upon vehicle or FIN treatment, as measured by quantitative RT-PCR analysis. The mRNA level of vehicle-treated cells was set to 1. n.s. , not significant. ( C ) Immunoblot analysis of EGFR in U373 (left) or T98G (right) cells after treatment with vehicle or FIN at the indicated concentration for 24 h. GAPDH was used as a loading control. The relative band intensities of the EGFR are shown below. The intensities of vehicle-treated samples were arbitrarily set to 1.

Article Snippet: Immunoblot assays were performed with antibodies against GAPDH (Cusabio, Houston, TX, USA, cat no. CSB-MA000184), β-catenin (Thermo Fisher Scientific, Waltham, MA, USA, cat no. MA1-300), phospho H3 at Ser 10 (Cell Signaling, Leiden, The Netherlands, cat no. 3377), phospho AKT (Thermo Fisher Scientific, Waltham, MA, USA, cat no. 700392), total AKT (Cell Signaling, Leiden, The Netherlands, cat no. 9272), SESN2 (Abcam, Cambridge, UK, cat no. ab178518), SOD2 (Cusabio, Houston, TX, USA, cat no. CSB-PA022398LA01HU), PRDX5 (Bethyl Laboratories, Montgomery, TX, USA, cat no. A305-339A), γH2AX (Novus Biologicals, Centennial, CO, USA, cat no. NB100-384), EGFR (Bioworld Technology, Bloomington, MN, USA, cat no. BS1533), P62 (Abcam, Cambridge, UK, cat no. ab56416), phospho P70S6K (Cell Signaling, Leiden, The Netherlands, cat no. 9205), total P70S6K (Cell Signaling, Leiden, The Netherlands, cat no. 9202), phospho 4EBP1 (Cell Signaling, Leiden, The Netherlands, cat no. 2855), and total 4EBP1 (Cell Signaling, Leiden, The Netherlands, cat no. 9644).

Techniques: Expressing, Transfection, Luciferase, Plasmid Preparation, Concentration Assay, Quantitative RT-PCR, Western Blot, Control

Journal: Pharmaceutics

Article Title: Antioxidant and Antiproliferative Activity of Finasteride against Glioblastoma Cells

doi: 10.3390/pharmaceutics13091410

Figure Lengend Snippet: High-dose finasteride shows negligible effect on DNA damage in glioblastoma cells. (A and B) U373 ( A ) and T98G ( B ) cells were treated with DMSO or temozolomide (TMZ) or 100 μM finasteride (FIN) for 24 h, and they were immunostained with a γH2AX antibody (red). Nuclear DAPI staining is presented in blue (left). The relative signal intensities of γH2AX are shown (right). Each dot means an individual cell. ( C ) Immunoblot analysis of γH2AX in U373 (left) or T98G (right) cells upon treatment of DMSO or TMZ or 100 μM FIN for 24 h. GAPDH was used as a loading control. The relative band intensities of γH2AX are represented. The intensities of vehicle-treated samples were arbitrarily set to 1. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Immunoblot assays were performed with antibodies against GAPDH (Cusabio, Houston, TX, USA, cat no. CSB-MA000184), β-catenin (Thermo Fisher Scientific, Waltham, MA, USA, cat no. MA1-300), phospho H3 at Ser 10 (Cell Signaling, Leiden, The Netherlands, cat no. 3377), phospho AKT (Thermo Fisher Scientific, Waltham, MA, USA, cat no. 700392), total AKT (Cell Signaling, Leiden, The Netherlands, cat no. 9272), SESN2 (Abcam, Cambridge, UK, cat no. ab178518), SOD2 (Cusabio, Houston, TX, USA, cat no. CSB-PA022398LA01HU), PRDX5 (Bethyl Laboratories, Montgomery, TX, USA, cat no. A305-339A), γH2AX (Novus Biologicals, Centennial, CO, USA, cat no. NB100-384), EGFR (Bioworld Technology, Bloomington, MN, USA, cat no. BS1533), P62 (Abcam, Cambridge, UK, cat no. ab56416), phospho P70S6K (Cell Signaling, Leiden, The Netherlands, cat no. 9205), total P70S6K (Cell Signaling, Leiden, The Netherlands, cat no. 9202), phospho 4EBP1 (Cell Signaling, Leiden, The Netherlands, cat no. 2855), and total 4EBP1 (Cell Signaling, Leiden, The Netherlands, cat no. 9644).

Techniques: Staining, Western Blot, Control