Journal: PLoS pathogens

Article Title: A cytoplasmic tail determinant in HIV-1 Vpu mediates targeting of tetherin for endosomal degradation and counteracts interferon-induced restriction.

doi: 10.1371/journal.ppat.1002609

Figure Lengend Snippet: Figure 4. Vpu ELV interacts and colocalizes with tetherin, and is incorporated into nascent virions. (A) 293T/tetherin cells were transfected twice over 48 h with siRNA oligonucleotide directed against UBAP1 or Non-targeting control. The cells were then infected with the indicated virus at an MOI of 2. 48 h later cell lysates were immunoprecipitated with an anti-tetherin monoclonal antibody. Lysates and immunoprecipitates were separated by SDS-PAGE and blotted for tetherin, UBAP1 or Vpu using LiCor quantitative Western blotting. Numbers under the Vpu lanes of the cell lysate represent relative band intensities. The histogram below the IP represents the ratio of tetherin band intensity to that of Vpu in the co-IP. (B) HeLa and 293T/tetherin cells were infected as in Figure 3 and stained for Vpu (green) and tetherin (red) and examined by confocal microscopy. Adjacent histograms quantify the degree of co-localization of Vpu ELV and tetherin (Pearson’s Correlation Coefficient calculated using ImageJ) for 20 individual cells. (C) and (D) 293T or 293T cells stably expressing tetherin delGPI were infected with HIV-1 wt, HIV-1 Vpu ELV or HIV-1 delVpu at an MOI of 1. 48 h post infection, cells were harvested and viral supernatants were pelleted through a 20% sucrose cushion. Cells and virions were subjected to SDS-PAGE and Western blotting for tetherin delGPI, Vpu, HIV-1 p24CA and Hsp90 and analyzed by LiCor quantitative imager. doi:10.1371/journal.ppat.1002609.g004

Article Snippet: Virion and cell lysates were then subjected to SDS-PAGE and Western blotted for HIV-1 p24CA (monoclonal antibody 183-H12-5C; kindly provided by B Chesebro through the NIH ARRP), rabbit anti-Hsp90 (Santa Cruz Biotechnologies), monoclonal mouse anti-HA.11 (Covance), polyclonal rabbit anti-HA (Rockland) and/or Vpu (rabbit polyclonal; kindly provided by K. Strebel through the NIH ARRP [67], and visualized by LiCor apparatus using fluorophores conjugated secondary antibodies (IRDye 800 Goat anti-rabbit, IRDye 680 Goat anti-mouse).

Techniques: Transfection, Control, Infection, Virus, Immunoprecipitation, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Staining, Confocal Microscopy, Stable Transfection, Expressing

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