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: Experimental and Therapeutic Medicine

Article Title: Effects of ozone stimulation of bronchial epithelial cells on proliferation and collagen synthesis of co-cultured lung fibroblasts

doi: 10.3892/etm.2018.6122

Figure Lengend Snippet: Representative microscopic images of HBECs and HLFs cultured alone or co-cultured with or without O 3 stimulation. (A) HBECs cultured alone without O 3 stimulation, (B) HBECs cultured alone with O 3 stimulation, (C) HLFs cultured alone, (D) HLFs co-cultured with HBECs without O 3 stimulation and (E) HLFs co-cultured with O 3 -stimulated HBECs (magnification, ×50). O 3 stimulation was given at a dosage of 2 ppm, 30 min for each treatment. Scale bar=100 µm. HBECs, human bronchial epithelial cells; HLFs, human lung fibroblasts; O 3 , ozone.

Article Snippet: Following this, cells were treated with TGF-β1 antibodies (1:1,000; cat. no. BA0290) at 37°C for 1–2 h and biotinylated goat anti-rabbit IgG antibodies (1:500; cat. no. BA1003; both Wuhan Boster Biological Technology Ltd.) at room temperature for 20 min. Reagent SABC (Wuhan Boster Biological Technology Ltd.) was added at room temperature for 20 min.

Techniques: Cell Culture

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of ozone stimulation of bronchial epithelial cells on proliferation and collagen synthesis of co-cultured lung fibroblasts

doi: 10.3892/etm.2018.6122

Figure Lengend Snippet: Time course of TGF-β1, TNF-α and PGE2 cytokine secretion in the supernatant of the co-culture system after human bronchial epithelial cells were stimulated ozone. TGF-β1 and TNF-α secretion was measured using ELISA and PGE2 secretion was measured using a radioimmunoassay. The levels of TGF-β1 remained high and continued to gradually increase for up to 24 h. TNF-α levels were relatively low and did not vary significantly over the 24 h. However, the secretion of PGE2 increased significantly between 6 and 12 h and thereafter plateaued until 24 h. Data are presented as the mean ± standard deviation (n=4). **P<0.01, *P<0.05 vs. 6 h. TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α; PGE2, prostaglandin E2.

Article Snippet: Following this, cells were treated with TGF-β1 antibodies (1:1,000; cat. no. BA0290) at 37°C for 1–2 h and biotinylated goat anti-rabbit IgG antibodies (1:500; cat. no. BA1003; both Wuhan Boster Biological Technology Ltd.) at room temperature for 20 min. Reagent SABC (Wuhan Boster Biological Technology Ltd.) was added at room temperature for 20 min.

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, RIA Assay, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of ozone stimulation of bronchial epithelial cells on proliferation and collagen synthesis of co-cultured lung fibroblasts

doi: 10.3892/etm.2018.6122

Figure Lengend Snippet: Effect of O 3 stimulation on the concentration of TGF-β1, TNF-α and PGE2 cytokines in HBECs cultured alone. The concentration levels of the cytokines TGF-β1 and TNF-α were measured using ELISA, and PGE2 was measured using a radioimmunoassay. The white bars represent HBECs cultured alone without O 3 -stimulation (control) and the filled bars represent HBECs cultured alone with O 3 -stimulation, respectively. Data are presented as the mean ± standard deviation (n=4). *P<0.05 vs. HBECS. TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α; PGE2, prostaglandin E2; O 3 , ozone.

Article Snippet: Following this, cells were treated with TGF-β1 antibodies (1:1,000; cat. no. BA0290) at 37°C for 1–2 h and biotinylated goat anti-rabbit IgG antibodies (1:500; cat. no. BA1003; both Wuhan Boster Biological Technology Ltd.) at room temperature for 20 min. Reagent SABC (Wuhan Boster Biological Technology Ltd.) was added at room temperature for 20 min.

Techniques: Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, RIA Assay, Control, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of ozone stimulation of bronchial epithelial cells on proliferation and collagen synthesis of co-cultured lung fibroblasts

doi: 10.3892/etm.2018.6122

Figure Lengend Snippet: Correlation between cytokine concentration and cell proliferation and collagen synthesis in HLFs co-cultured with O 3 -stressed HBECs. (A and B) A positive linear correlation between proliferation of HLFs and the concentration of TGF-β1 in the co-culture supernatant. Collagen synthesis capacity in the culture supernatant was also positively correlated with TGF-β1 concentration (r=0.758, P=0.018). (C and D) There was no correlation between the proliferation and TNF-α concentration (r=0.209, P=0.589), nor between collagen synthesis and TNF-α concentration (r=0.311, P=0.415). (E and F) A negative linear correlation between the proliferation and PGE2 concentration of HLFs (r=0.783, P=0.013) and between collagen synthesis and PGE2 concentration (r=0.817, P=0.007) was indicated. TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α; PGE2, prostaglandin E2; O 3 , ozone; HBECs, human bronchial epithelial cells; HLFs, human lung fibroblasts; OD, optical density.

Article Snippet: Following this, cells were treated with TGF-β1 antibodies (1:1,000; cat. no. BA0290) at 37°C for 1–2 h and biotinylated goat anti-rabbit IgG antibodies (1:500; cat. no. BA1003; both Wuhan Boster Biological Technology Ltd.) at room temperature for 20 min. Reagent SABC (Wuhan Boster Biological Technology Ltd.) was added at room temperature for 20 min.

Techniques: Concentration Assay, Cell Culture, Co-Culture Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of ozone stimulation of bronchial epithelial cells on proliferation and collagen synthesis of co-cultured lung fibroblasts

doi: 10.3892/etm.2018.6122

Figure Lengend Snippet: Representative images and corresponding quantitative expression level of TGF-β1 in HBECs with or without O 3 stimulation. HBECs were cultured alone without O 3 stimulation (control) or cultured alone with O 3 stimulation. (A) TGF-β1 in HBECs was labeled by immunocytochemistry and imaged by photomicroscope. (B) Expression level of TGF-β1 in HBECs was quantified according to the averaged photodensity of the images. Data are presented as the mean ± standard deviation (n=4). *P<0.05 vs. HBECs. HBECs, human bronchial epithelial cells; HLFs, human lung fibroblasts; O 3 , ozone; TGF-β1, transforming growth factor-β1.

Article Snippet: Following this, cells were treated with TGF-β1 antibodies (1:1,000; cat. no. BA0290) at 37°C for 1–2 h and biotinylated goat anti-rabbit IgG antibodies (1:500; cat. no. BA1003; both Wuhan Boster Biological Technology Ltd.) at room temperature for 20 min. Reagent SABC (Wuhan Boster Biological Technology Ltd.) was added at room temperature for 20 min.

Techniques: Expressing, Cell Culture, Control, Labeling, Immunocytochemistry, Standard Deviation