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

Journal: Molecular Cancer Research

Article Title: The Opposing Effect of Hypoxia-Inducible Factor-2α on Expression of Telomerase Reverse Transcriptase

doi: 10.1158/1541-7786.mcr-07-0065

Figure Lengend Snippet: FIGURE 4. HIF-2a occupancy on the hTERT promoter, recruitment of p300, and changes in histone H3 acetylation in A498 and U251 cells under hypoxic conditions. A. Schematic presentation of the hTERT promoter and locations of the ChIP PCR amplicons relative to the translational start codon (ATG). The putative HREs in region a are indicated (16). Region b was also found to interact with HIF-1a according to Anderson et al. (10). B. The ChIP assay for the presence of HIF-2a, p300, and histone H3 acetylation at the hTERT promoter in hypoxia-treated A498 and U251 cells. The precipitated DNA was subject to PCR analysis using the primer pairs specific to the hTERT promoter regions a and b. Input is sonicated chromatin without immunoprecipitation, but reverse cross-linked and purified together with ChIP samples. HRE, hypoxia-responsive element; H and N, the cells were cultured under hypoxic and normal oxygen conditions, respectively. Ab, no antibody controls. H3-Ac, the antibody against acetylated histone H3.

Article Snippet: The membranes were probed with the specific mouse monoclonal antibody against HIF-1a or HIF-2a rabbit polyclonal antibody (Novus Biologicals), followed by a secondary anti-mouse or rabbit horseradish peroxidase–conjugated immunoglobulin G, and developed with the enhanced chemiluminescence method (Amersham).

Techniques: Sonication, Immunoprecipitation, Purification, Cell Culture

Journal: Biology open

Article Title: New K50R mutant mouse models reveal impaired hypusination of eif5a2 with alterations in cell metabolite landscape.

doi: 10.1242/bio.059647

Figure Lengend Snippet: Fig. 2. Generation of eif5a1-K50R and eif5a2-K50R mutant mice. Targeting strategy and sequencing results for eif5a1-K50R (A), eif5a2-K50R (B) and eif5a2-ΔK50-G52 (C) mouse lines. In schematics of exon structure, filled rectangles denote coding exons, and empty exons correspond to untranslated regions. Relative position of ATG (start codon) and K50R codon are indicated, with target site at K50 magnified to show the position of flanking amino acids and DNA sequence (left panels). DNA sequence highlighted in bold – K50 codon, red – R50 mutation, green – PAM of gRNA used for CRISPR targeting. Sanger sequencing chromatograms (right panels) of the wild-type allele and K50R-mutated allele in a heterozygous (A) and homozygous (B and C) format are labeled with asterisks to denote mutated nucleotides, and with brackets marking K50R codon position. PAM, protospacer adjacent motif; WT, wild-type.

Article Snippet: The mouse anti-eIF5A2 antibody (TA505100, Origene) was tested to be specific for eIF5A2 protein only and did not detect eIF5A1 protein by western blot when using commercially available HEK293 cell lysates that overexpress either eIF5A1 or eIF5A2 (LC419616 or LC412495, Origene) (Fig. S5).

Techniques: Mutagenesis, Sequencing, CRISPR, Labeling

Journal: Biology open

Article Title: New K50R mutant mouse models reveal impaired hypusination of eif5a2 with alterations in cell metabolite landscape.

doi: 10.1242/bio.059647

Figure Lengend Snippet: Fig. 3. Validation of the genotype and hypusination status of the eif5a2-K50R mouse model. (A) Mice were genotyped to confirm the eif5a2-K50R mutation using mouse tail DNA preps. The O208/O209 primer pair was used for PCR resulting in a 503 bp product, which was digested using PflFI. eif5a2- K50R mutant PCR product is cut into two bands, while the wild-type product is not digested. (B) Protein lysates from brain tissue of the same mice were used to detect hypusinated eif5a2 levels. Brain tissue was used because of its high endogenous eif5a2 expression. As expected, eif5a2K50R/K50R mice lack hypusinated eif5a2. (C) Quantification of eif5a2 levels relative to β-actin in (B). * indicates a statistically significant difference in eif5a2 protein levels in wild- type and eif5a2-K50R mouse brain lysates (P=0.0194). WT, wild-type.

Article Snippet: The mouse anti-eIF5A2 antibody (TA505100, Origene) was tested to be specific for eIF5A2 protein only and did not detect eIF5A1 protein by western blot when using commercially available HEK293 cell lysates that overexpress either eIF5A1 or eIF5A2 (LC419616 or LC412495, Origene) (Fig. S5).

Techniques: Biomarker Discovery, Mutagenesis, Expressing

Journal: Biology open

Article Title: New K50R mutant mouse models reveal impaired hypusination of eif5a2 with alterations in cell metabolite landscape.

doi: 10.1242/bio.059647

Figure Lengend Snippet: Fig. 4. eif5a1-eif5a2 mRNA expression in mouse tissue. The eif5A1 and eif5A2 mRNA tissue expression patterns were analyzed using the mouse ENCODE database that profiles mouse embryonic and adult tissues using RNA sequencing through the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). mRNA levels show that eif5a1 is almost universally expressed in all tissues investigated, whereas eif5a2 shows a much more restricted expression pattern, at around 30-50 times lower levels. Absolute mRNA levels are expressed as reads per kilobase per million (RKPM).

Article Snippet: The mouse anti-eIF5A2 antibody (TA505100, Origene) was tested to be specific for eIF5A2 protein only and did not detect eIF5A1 protein by western blot when using commercially available HEK293 cell lysates that overexpress either eIF5A1 or eIF5A2 (LC419616 or LC412495, Origene) (Fig. S5).

Techniques: Expressing, RNA Sequencing

Journal: Biology open

Article Title: New K50R mutant mouse models reveal impaired hypusination of eif5a2 with alterations in cell metabolite landscape.

doi: 10.1242/bio.059647

Figure Lengend Snippet: Fig. 5. eIF5A1-eIF5A2 gene aberrations in human cancer. An overview of eIF5A1 and eIF5A2 gene aberrations in human cancer was obtained using the cBioPortal website for Cancer Genomics (https://www.cbioportal.org/). Quantitative analysis of eIF5A1/2 genetic abnormalities using the 32 TCGA Pan Cancer studies, with 10,967 samples representing most major human cancer types. Analyses was performed using website default settings.

Article Snippet: The mouse anti-eIF5A2 antibody (TA505100, Origene) was tested to be specific for eIF5A2 protein only and did not detect eIF5A1 protein by western blot when using commercially available HEK293 cell lysates that overexpress either eIF5A1 or eIF5A2 (LC419616 or LC412495, Origene) (Fig. S5).

Techniques:

Journal: Biology open

Article Title: New K50R mutant mouse models reveal impaired hypusination of eif5a2 with alterations in cell metabolite landscape.

doi: 10.1242/bio.059647

Figure Lengend Snippet: Fig. 7. Metabolomics of primary mouse fibroblasts harboring eif5a2-K50R mutation reveal broad metabolic changes. (A) A heatmap of significantly differentially abundant metabolites between wild-type and eif5a2K50R/K50R-derived fibroblasts. (B) A volcano plot reveals that 47 metabolites are differentially abundant, with 41 metabolites showing increased levels (FDR<0.05) in K50R cells. Within these significantly elevated metabolites, interesting patterns emerged with glutathione metabolism (C), and vitamins and cofactors (D). Data are expressed as mean±s.e. GSH, glutathione; NAD, nicotinamide adenine dinucleotide; FAD, flavin adenine dinucleotide; CoA, coenzyme A. WT, wild-type.

Article Snippet: The mouse anti-eIF5A2 antibody (TA505100, Origene) was tested to be specific for eIF5A2 protein only and did not detect eIF5A1 protein by western blot when using commercially available HEK293 cell lysates that overexpress either eIF5A1 or eIF5A2 (LC419616 or LC412495, Origene) (Fig. S5).

Techniques: Mutagenesis, Derivative Assay