Journal: Nature biotechnology

Article Title: Efficient integration of heterogeneous single-cell transcriptomes using Scanorama

doi: 10.1038/s41587-019-0113-3

Figure Lengend Snippet: Scanorama correctly integrates a simple collection of datasets where other methods fail. (a) We apply Scanorama to a collection of three datasets17: one entirely of Jurkat cells (n = 3257 cells) (Experiment 1), one entirely of 293T cells (n = 2885 cells) (Experiment 2), and a 50:50 mixture of Jurkat and 293T cells (n = 3388 cells) (Experiment 3). (b) Our method correctly identifies Jurkat cells (orange) and 293T cells (blue) as two separate clusters. (c,d) Existing methods for scRNA-seq dataset integration are sensitive to the order in which they consider datasets (see Supplementary Fig. 1) and can incorrectly merge a Jurkat dataset and a 293T dataset together first before subsequently incorporating a 293T/Jurkat mixture, forming clusters that do not correspond to actual cell types.

Article Snippet: Panorama of 293Tand Jurkat cells We obtained three separate datasets consisting of 293T cells, Jurkat cells, and a 50:50 mixture of 293T and Jurkat cells from 10x Genomics 17 .

Techniques:

Journal: PLoS Pathogens

Article Title: Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

doi: 10.1371/journal.ppat.1009246

Figure Lengend Snippet: ( A) Schematic illustration of SARS-CoV-2 S including receptor binding domain (RBD) in green and proteolytic cleavage sites (S1/S2, S2’). Amino acid sequences around the S1/S2 recognition sites of SARS-CoV-2 S are indicated while the multibasic site is highlighted in purple. Amino acid mutations are highlighted in light blue while deletions are marked with orange dashes. (B) Overall structure of the SARS-CoV-2 S protein (PDB: 6VYB). RBD core is shown in green. Pro-Arg-Arg-Ala-Arg residues are shown in yellow. (C,D) Representative western blots of HIV Pseudoviruses (C) and Virus Like Particles (VLPs) (D) harbouring the indicated SARS-CoV-2 S protein mutants (detected with anti-S antibody) and produced in 293-wt and 293T-ΔFURIN cells. Expression of HIV capsid protein (p24) (C) and SARS-CoV-2 nucleoprotein (D) is shown as loading control. (E) Representative western blot analysis of spike and nucleoprotein present in SARS-CoV-2 viral particles produced in 293T-hACE2 and 293T-hACE2-ΔFURIN after 42 hours post infection. The cleaved S in (C) (D) and (E) identifies the S2 subunit.

Article Snippet: For the generation of hACE2 over-expressing 293T cells, the human hACE2 ORF was PCR amplified from Addgene plasmid 1786 and C-terminally fused with the porcine teschovirus-1-derived P2A cleavage sequence (ATNFSLLKQAGDVEENPGP) followed by the blasticidin resistance gene.

Techniques: Binding Assay, Western Blot, Virus, Produced, Expressing, Control, Infection

Journal: PLoS Pathogens

Article Title: Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

doi: 10.1371/journal.ppat.1009246

Figure Lengend Snippet: (A) Infection of 293T-hACE2 cells with GFP expressing HIV pseudotyped with SARS-CoV-2 S mutants, measured as proportion of cell area expressing GFP. Viruses were produced in either 293T-wt or 293T-ΔFURIN cells. (B) and (C) Infection data of 293T-hACE2 cells with HIV pseudotyped with SARS-CoV-2 S mutants as in (A), with cells pre-treated for 2 hours with either DMSO or 25μM lysosomal inhibitor E64d as indicated. Statistical analysis was performed using Student t -test * P< 0.05; ** P< 0.01, “ns” not significant. (D) Representative western blot of viral particles produced from SARS-CoV-2 infection of 293T-hACE2 and 293T-hACE2-ΔFURIN cells at 72 hours post infection. Spike and nucleoprotein are detected (left panel). Total protein content of virus preparation by Coomassie staining (right panel). (E) Plaque assay of Vero-hACE2-TMPRSS2 infected with viruses produced in D. (F) and (G) Immunofluorescence images displaying infection of Caco2 BVDV-Npro cells (F) and 293T-hACE2 (G) with equalised amounts of SARS-CoV-2 virus produced in 293T-hACE2 and 293T-hACE2-ΔFURIN cells. Spike (green) and nuclei (blue) are shown. Scale bar, 50 μm. (H) and (I) Quantification of SARS-CoV-2 infected cells shown in (F) and (G). * P< 0.05; ***, P< 0.001 analysed using Student t -test. Data are expressed as mean +/- SEM (n = 2).

Article Snippet: For the generation of hACE2 over-expressing 293T cells, the human hACE2 ORF was PCR amplified from Addgene plasmid 1786 and C-terminally fused with the porcine teschovirus-1-derived P2A cleavage sequence (ATNFSLLKQAGDVEENPGP) followed by the blasticidin resistance gene.

Techniques: Infection, Expressing, Produced, Western Blot, Virus, Staining, Plaque Assay, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Interferon regulatory factor 8 regulates caspase-1 expression to facilitate Epstein-Barr virus reactivation in response to B cell receptor stimulation and chemical induction

doi: 10.1371/journal.ppat.1006868

Figure Lengend Snippet: A. Schematic representation of the promoter of human CASP1 . IRF8 consensus binding site is highlighted in green. The ATG of CASP1 is highlighted in red. B. The pGL2-CASP1p constructs (with or without IRF8 consensus site) and the IRF8 consensus site mutated construct were co-transfected into 293T cells with either vector control or IRF8 expression vectors. Luciferase assays were performed 36 hrs post-transfection. The value of cells transfected with empty vectors was set as 1. The results were presented as mean ± standard deviation of triplicate assays. C. The pGL2-CASP1p1 construct was co-transfected into 293T cells with either vector control, wild-type IRF8 (WT) or IRF8 DNA binding mutant (K108E) expression vectors and luciferase assays were performed 36 hrs post-transfection. The value of cells transfected with empty vectors was set as 1. The results were presented as mean ± standard deviation of triplicate assays. D. The pGL2-CASP1p1 construct was co-transfected into 293T cells with either vector control or IRF8 and IRF1 expression vectors and luciferase assays were performed 36 hrs post-transfection. The value of cells transfected with empty vectors was set as 1. The results were presented as mean ± standard deviation of triplicate assays. *** p<0.001.

Article Snippet: 293T cells (a gift from Diane Hayward, Johns Hopkins University) were grown in DMEM media supplemented with 10% FBS.

Techniques: Binding Assay, Construct, Transfection, Plasmid Preparation, Control, Expressing, Luciferase, Standard Deviation, Mutagenesis

Journal: PLoS Pathogens

Article Title: Interferon regulatory factor 8 regulates caspase-1 expression to facilitate Epstein-Barr virus reactivation in response to B cell receptor stimulation and chemical induction

doi: 10.1371/journal.ppat.1006868

Figure Lengend Snippet: A. Caspase-1 depletion suppresses KAP1 degradation. Protein extracts form were analyzed by western blot using antibodies against KAP1, PAX5, DNMT3A and Caspase-8 (CASP8). B. Caspase-1 and -8 cleave KAP1 in vitro . HA-KAP1 and the antibody recognition sites are labeled as indicated. HA-tagged KAP1 was immuoprecipitated from transfected 293T cells using HA magnetic beads. The beads-bound HA-KAP1 was incubated with individual recombinant caspase for 2 hrs at 37°C. WB was performed using either anti-HA or anti-KAP1 antibodies. The relative positions of cleaved fragments were labeled as indicated.

Article Snippet: 293T cells (a gift from Diane Hayward, Johns Hopkins University) were grown in DMEM media supplemented with 10% FBS.

Techniques: Western Blot, In Vitro, Labeling, Transfection, Magnetic Beads, Incubation, Recombinant

Journal: Nature methods

Article Title: Kilohertz two-photon brain imaging in awake mice

doi: 10.1038/s41592-019-0597-2

Figure Lengend Snippet: (a) Conventional epi-fluorescence image in motor cortex of an anesthetized mouse after intravenous administration of rhodamine-B. Dashed box encloses the artery in b . (b) Image time series (2.5 ms per frame; 200 kHz laser pulse rate; 2.2 mW per beamlet) taken by high-speed two-photon imaging reveals flow of injected, fluorescent HEK-293 cells. Arrowheads mark a cell’s progress. (c) Flow speed map for the vessel in b . (d) Trajectories of individual HEK-293 cells. Each trajectory is encoded in color and superposed on an epi-fluorescence image of the vasculature. (e) Flow speeds in neocortical arteries had a parabolic cross-sectional profile. For 15 different cross-sections chosen within 3 different arteries (>50 μm in diameter; N = 3 mice), we computed flow speeds, V ( r ), as a function of the radial deviation, r , from the vessel’s longitudinal axis. We fit (red curve) the data (blue points) to V/V max = 1 – ( r/R ) n , where V max is each vessel’s peak flow speed and R is its radius. The fitting parameter, n = 2.0 ± 0.3 (95% C.I.), revealed the flow speed’s quadratic profile. Error bars: s.e.m. ( N = 15 cross-sections). (f) Flow speed map determined by 1-kHz-two-photon imaging (200 kHz laser repetition rate; 2.9 mW per beamlet; 450 × 110 μm 2 field of view). (g) Trajectories of individual HEK-293 cells, determined from the same dataset used for f . (h) Periodic fluctuations in blood flow, as computed within the encircled area in f . The heart rate of ~150 beats·min −1 determined by high-speed imaging matches conventional measurements in ketamine-xylazine-anesthetized mice . (i) Sketch of the mouse superior sagittal sinus (SSS). Inset : We targeted areas near bregma for imaging (dotted rectangle). (j) Example time traces of bridging vein diameter determined by 200-Hz-imaging (450 × 300 μm 2 field of view; 200 kHz laser pulse rate; 2.2 mW per beamlet) after intravenous injection of rhodamine-B. During wakefulness, the vein diameter (blue trace) exhibited fast constriction (red dots) and dilation (cyan dots), which anesthesia abolished (gray trace). (k) Negative- and positive-going changes in vein diameter, relative to each vessel’s mean diameter, during constriction (red curve) and dilation (blue curve) in 4 awake mice. Constriction and dilation rates were, respectively, −6.5 ± 0.9% and +4.0 ± 0.7% per 100 ms (mean ± s.e.m; 36 events of each type). Shading: s.e.m. Scale bars: 50 μm in a – d, f , g .

Article Snippet: We incubated the samples at 37°C for 1–2 h and washed the quantum-dot-labeled HEK-293T cells twice with phosphate buffered saline (PBS); after each wash we collected the cells by centrifugation at 200 G-force for 5 min. We re-suspended the final cell pellet in PBS at a concentration of ~2.5 · 10 7 cells/mL.

Techniques: Fluorescence, Imaging, Injection