Journal: Journal of Virology

Article Title: Innate Immune Response to Influenza Virus at Single-Cell Resolution in Human Epithelial Cells Revealed Paracrine Induction of Interferon Lambda 1

doi: 10.1128/JVI.00559-19

Figure Lengend Snippet: MOI-dependent temporal expression dynamics of the NS1 protein (PR8-GFP virus) in human respiratory epithelial cells (A549 H2B-mCherry) using time-lapse microscopy. Green indicates NS1 expression (GFP). The cell nuclei (H2B-mcherry) are depicted in red. (A) Time course from 3 to 12 hpi. One representative field is shown for each MOI. (B) Quantification of the number of GFP-positive cells for the four MOIs used (approximately 25,000 cells/well). For MOI of 0.2, 0.6, and 2, averages of two replicate wells ± SDs are shown. For infection at an MOI of 10 and mock infection, data from one single well are shown. (C) Heat map showing the median of fluorescence intensity of the infected cells (GFP+ cells). (D) Table showing the measured percentages of infected cells at 12 hpi, as well as the theoretical percentages of cells infected either with ≥1 virion (all infected cells) or >1 virion (multiple infections).

Article Snippet: A549 H2B-mCherry-infected or mock-infected cells were detached from the cell culture plates using trypsin-0.05% EDTA (Gibco), washed with cell culture media, and passed through a 40-μm cell strainer to prepare single-cell suspensions, and the final concentration was set at 1,000 cells/μl.

Techniques: Expressing, Virus, Time-lapse Microscopy, Infection, Fluorescence

Journal: Journal of Virology

Article Title: Innate Immune Response to Influenza Virus at Single-Cell Resolution in Human Epithelial Cells Revealed Paracrine Induction of Interferon Lambda 1

doi: 10.1128/JVI.00559-19

Figure Lengend Snippet: Stimulation of A549 cells with TNF-α, IFN beta, IFNL1 (100 U/ml), or combinations of those cytokines or infected with PR8-GFP (MOI, 2) for 8 h. Induction of IFNL1 was not detected upon stimulation with those cytokines by qRT-PCR. Gene expression of the indicated genes (IFNs, with Mx1 and CCL5 to confirm the effectiveness of the treatments) was analyzed. Averages of biological triplicates ± SDs are shown.

Article Snippet: A549 H2B-mCherry-infected or mock-infected cells were detached from the cell culture plates using trypsin-0.05% EDTA (Gibco), washed with cell culture media, and passed through a 40-μm cell strainer to prepare single-cell suspensions, and the final concentration was set at 1,000 cells/μl.

Techniques: Infection, Quantitative RT-PCR, Gene Expression

Journal: Molecular Biology of the Cell

Article Title: Mps1 regulates spindle morphology through MCRS1 to promote chromosome alignment

doi: 10.1091/mbc.E18-09-0546

Figure Lengend Snippet: MCRS1 S65 phosphorylation-deficient mutant exhibits a defect in chromosome segregation. (A) Time-lapse live-cell images. A549 cells expressing H2B-CFP were transfected with MCRS1 siRNA. Cells were then arrested at G2 by RO3306 and released into fresh medium for monitoring chromosome segregation. Time after nuclear envelope breakdown was counted by minutes. Misaligned chromosomes in MCRS1 siRNA cells were magnified in a rectangle. Bar: 5 μm. (B) Mitotic duration of MCRS1 depleted cells. Results from three independent experiments of panel A were combined and are shown together as mean ± SEM (total 120 mitotic cells). Two-tailed P value was calculated by unpaired Student’s t test. **** means P value is less than 0.0001, *** less than 0.001, and ** less than 0.01. NS stands for not significant. (C) Immunofluorescence of mitotic cells. HeLa Tet-on cells stably expressing MCRS1-GFP were fixed and processed for immunofluorescence using GFP antibody. DNA was stained by Hoechst 33342. Bar: 5 μm. (D) Stable cell line expressing MCRS1-GFP WT, S65A, or S65D. HeLa Tet-on cells were transfected with MCRS1 siRNA and processed for Western blot to examine MCRS1 protein level. (E) Mitotic duration of stable cell lines. Stable cell lines were transfected with siRNA and mitotic duration was counted by live-cell DIC microscope movie. Results from two independent experiments counting ∼40 cells were combined and are shown here as mean ± SEM. Two-tailed P value was calculated similarly as in panel B. (F) Abnormal chromosome alignment at metaphase. Stable cell lines were transfected with siRNA and treated with MG132 for 1 h. Metaphase cells were counted either normal or abnormal, based on DNA and mitotic spindle distribution. Results from three independent experiments counting ∼60 cells each were combined and are shown here as mean ± SEM. Two-tailed P value was calculated similarly as in panel B. Bar: 5 μm.

Article Snippet: Fluorescence time-lapse imaging of A549 cells expressing CFP-H2B was recorded every 10 min for a total duration of 24 h with a 10× objective in a Nikon Eclipse Ti-E microscope equipped with a temperature- and CO 2 -controlled stage incubation unit (Okolab).

Techniques: Mutagenesis, Expressing, Transfection, Two Tailed Test, Immunofluorescence, Stable Transfection, Staining, Western Blot, Microscopy