Journal: Journal of Virology

Article Title: Exacerbated Apoptosis of Cells Infected with Infectious Bursal Disease Virus upon Exposure to Interferon Alpha

doi: 10.1128/JVI.00364-18

Figure Lengend Snippet: IFN treatment triggers apoptosis of IBDV-infected HeLa cells. HeLa cells mock infected or infected with IBDV (MOI of 2) were treated with hIFN-α (1,000 IU/ml) at 3, 6, 9, or 12 h p.i. (samples named throughout the text M+3, M+6, M+9, and M+12 and I+3, I+6, I+9, and I+12, respectively), as indicated, or remained untreated (−) (named throughout the text M and I, respectively). Cells were analyzed at 24 h p.i. by using different assays. (A) Phase-contrast microscopy. (B) Fluorescence microscopy after incubation with the Live/Dead cell imaging reagent to discriminate live (green) from dead (red) cells. (C) Western blot analysis of cells mock infected or infected with IBDV and treated with hIFN-α at the indicated times p.i. with different antibodies: anti-PARP, anti-total PKR (t-PKR), anti-phosphorylated (Thr446) PKR (p-PKR), anti-total eIF2α (t-eIF2α), anti-phosphorylated (Ser52) eIF2α (p-eIF2α), anti-Mx, anti-ISG-56, and anti-VP3. The PARP cleavage product is denoted c-PARP. Antibodies to β-actin were used for a protein loading control. (D) Apoptosis was measured from duplicate samples by using the Caspase-Glo 3/7 assay kit, and each determination was carried out in duplicate. Caspase values from infected cell samples were normalized to those from mock-infected cells. Bars indicate means ± standard deviations based on duplicate samples from two independent experiments. Striped bars, infected cell samples not treated with IFN; solid bars, infected cell samples treated with IFN. (E) Analysis of the IFN dose-response effect on caspase 3/7 activity. Mock-infected or infected cells were treated with increasing doses (10-fold) of hIFN-α (from 1 to 105 IU/ml), and apoptosis was measured at 24 h p.i. by using the Caspase-Glo 3/7 assay kit. (F) Comparative analysis of apoptosis levels in IBDV-infected HeLa cells treated with IFN (1,000 IU/ml) at 3 h p.i. and uninfected cells treated with different doses of staurosporine (STS) (0.5, 1, and 2 μM), collected at 24 h p.i. or posttreatment, respectively. * and ** indicate P values of <0.05 and <0.01, respectively as determined by unpaired Student's t test.

Article Snippet: After 1 h of virus adsorption at 37°C, the medium was removed and replaced with fresh DMEM supplemented with 2% FCS and the IncuCyte caspase 3/7 apoptosis assay reagent (Essen BioScience), according to the manufacturer's instructions.

Techniques: Infection, Microscopy, Fluorescence, Incubation, Imaging, Western Blot, Caspase-Glo Assay, Activity Assay

Journal: Journal of Virology

Article Title: Exacerbated Apoptosis of Cells Infected with Infectious Bursal Disease Virus upon Exposure to Interferon Alpha

doi: 10.1128/JVI.00364-18

Figure Lengend Snippet: Triggering of apoptosis during IBDV infection in cells treated with IFN correlates with PKR phosphorylation and upregulation of IFN-β gene expression. HeLa cells mock infected (M) or infected with IBDV (MOI of 2) (I) were treated or not with hIFN-α (1,000 IU/ml of at 3 h p.i.) (M+3 and I+3, respectively) and harvested at the indicated times p.i. (A) Western blot analysis of total cell extracts with anti-PARP, anti-t-PKR, anti-p-PKR, and anti-VP3 antibodies. The Western blot corresponding to β-actin was used as a protein loading control. The PARP cleavage product is denoted c-PARP. (B) Real-time quantitative cell death analysis. Mock-infected and IBDV-infected HeLa cells treated as indicated above were incubated with the IncuCyte caspase-3/7 apoptosis assay reagent as described in Materials and Methods, and cell cultures were monitored with an IncuCyte Zoom system apparatus during 24 after infection. Apoptotic cell death in duplicate cultures under each experimental condition, visualized by the appearance of green cells, was analyzed with IncuCyte Zoom software. (C to E) RT-qPCR analysis of IFN-β (C) and viral (D and E) RNAs during infection in cells treated with hIFN-α. In panel E, cells were treated or not with 7DMA (0.2 mM) after virus adsorption and then treated (I+3) (solid bars) or not (I) (striped bars) with hIFN-α at 3 h p.i. The expression levels of the selected genes, as indicated, were determined by SYBR green-based RT-qPCR. The expression level of the IFN-β target gene was normalized to the HPRT mRNA content and is presented on a log10 scale as a fold change over the level in mock-infected HeLa cells. (F) Apoptosis in samples from M, M+3, I, and I+3 cells treated or not treated with 7DMA (0.2 mM) after virus adsorption was measured at 24 h p.i. by using the Caspase-Glo 3/7 assay kit. Each determination was carried out in duplicate. The presented data correspond to the means ± the standard deviations of results from two independent experiments. The caspase values for infected cell samples were normalized to those for mock-infected cells. Bars indicate means ± standard deviations based on data from duplicate samples. * and ** indicate P values of <0.05 and <0.01, respectively, as determined by unpaired Student's t test.

Article Snippet: After 1 h of virus adsorption at 37°C, the medium was removed and replaced with fresh DMEM supplemented with 2% FCS and the IncuCyte caspase 3/7 apoptosis assay reagent (Essen BioScience), according to the manufacturer's instructions.

Techniques: Infection, Expressing, Western Blot, Incubation, Apoptosis Assay, Software, Quantitative RT-PCR, Virus, Adsorption, SYBR Green Assay, Caspase-Glo Assay

Journal: Journal of Virology

Article Title: Exacerbated Apoptosis of Cells Infected with Infectious Bursal Disease Virus upon Exposure to Interferon Alpha

doi: 10.1128/JVI.00364-18

Figure Lengend Snippet: IFN treatment triggers apoptosis of IBDV-infected chicken DF-1 cells. DF-1 cells were infected with IBDV (MOI of 2) and treated or not with 1,000 U/ml of chIFN-α at 3 h p.i. Mock-infected (M and M+3) and IBDV-infected (I and I+3) cells were analyzed at 16 h p.i. (A) Cells were incubated with the IncuCyte caspase 3/7 apoptosis assay reagent, and images were recorded with an IncuCyte Zoom system apparatus at 16 h p.i. Apoptotic cells are green. (B) Apoptosis measurement. As a control for apoptosis, uninfected DF-1 cells were treated with different doses of staurosporine (0.5, 1, and 2 μM) for 16 h. Apoptosis in duplicate samples was measured by using the Caspase-Glo 3/7 assay kit, and each determination was carried out in duplicate. Caspase values for infected cell samples were normalized to those for mock-infected cells. Bars indicate means ± standard deviations based on data from duplicate samples from two independent experiments. ** indicates a P value of <0.01, as determined by unpaired Student's t test.

Article Snippet: After 1 h of virus adsorption at 37°C, the medium was removed and replaced with fresh DMEM supplemented with 2% FCS and the IncuCyte caspase 3/7 apoptosis assay reagent (Essen BioScience), according to the manufacturer's instructions.

Techniques: Infection, Incubation, Apoptosis Assay, Caspase-Glo Assay

Journal: Cell Metabolism

Article Title: Brain-Sparing Sympathofacilitators Mitigate Obesity without Adverse Cardiovascular Effects

doi: 10.1016/j.cmet.2020.04.013

Figure Lengend Snippet:

Article Snippet: Protein Assay Dye Reagent Concentrate , Bio-Rad , Cat#5000006.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Quantitation Assay, SYBR Green Assay, Reverse Transcription, Software, Fluorescence, Imaging, High Throughput Screening Assay, Mass Spectrometry

Journal: Cells

Article Title: Widespread Distribution of Luteinizing Hormone/Choriogonadotropin Receptor in Human Juvenile Angiofibroma: Implications for a Sex-Specific Nasal Tumor

doi: 10.3390/cells13141217

Figure Lengend Snippet: LHCGR is enriched in cells located around blood vessels. Examples show confocal fluorescence images of RNAscope in situ hybridization in JA tissue samples using a probe specifically directed against LHCGR . ( A – C ) LHCGR expression (red) is primarily restricted to cells surrounding the blood vessels but also scattered in the tissue between blood vessels ( A , C ). In addition to the empty dark vascular lumen, blood vessels can be identified by DAPI (4′,6-diamidino-2-phenylindole, blue) nuclear staining outlining their structure ( B ). An overlay of the two images (merged, C ) summarizes the data of patient #1754 (17 y). Blood vessels are marked with an asterisk.

Article Snippet: After hybridization (2 h, 40 °C), sections were rinsed twice for 2 min in wash buffer (ACD Bio-Techne), sequentially treated with v2-amplification reagents at 40 °C (30 min Amp1, 30 min Amp2, 15 min Amp3, and 15 min HRP-C1), and incubated in tyramide-amplification-based fluorescence reagent (15 min, 40 °C TSA Vivid Dye 650, Tocris Cat. No. 5748, Bristol, UK) with intermittent washes at room temperature between reagents.

Techniques: Fluorescence, RNAscope, In Situ Hybridization, Expressing, Staining

Journal: Cells

Article Title: Widespread Distribution of Luteinizing Hormone/Choriogonadotropin Receptor in Human Juvenile Angiofibroma: Implications for a Sex-Specific Nasal Tumor

doi: 10.3390/cells13141217

Figure Lengend Snippet: ( A – O ) LHCGR RNAscope performed on JA tissue sections from 5 patients. Confocal fluorescence images of JA tissue from three patients showing representative views of the widespread LHCGR distribution ( A , D , G , J , M ). The dashed white boxes are shown in ( B , E , H , K , N ) as merged images of LHCGR expression and DAPI labeling from the vascular regions. White arrows indicate the nuclei magnified in ( C , F , I , L , O ) showing the distribution of RNA puncta above and next to the nuclei. Blood vessels are marked with an asterisk.

Article Snippet: After hybridization (2 h, 40 °C), sections were rinsed twice for 2 min in wash buffer (ACD Bio-Techne), sequentially treated with v2-amplification reagents at 40 °C (30 min Amp1, 30 min Amp2, 15 min Amp3, and 15 min HRP-C1), and incubated in tyramide-amplification-based fluorescence reagent (15 min, 40 °C TSA Vivid Dye 650, Tocris Cat. No. 5748, Bristol, UK) with intermittent washes at room temperature between reagents.

Techniques: RNAscope, Fluorescence, Expressing, Labeling