Journal: Journal of Infection and Public Health

Article Title: A three-dimensional A549 cell culture model to study respiratory syncytial virus infections

doi: 10.1016/j.jiph.2020.03.011

Figure Lengend Snippet: RSVA infection and mucin production in A549 spheroids. A549 Spheroids were inoculated with 1 MOI of RSVA 2001/2-20 and incubated for 3 (3A) and 7 (3B ) days. At the assigned time-points, spheroids were immunostained with anti-RSV F antibody (green), anti-MUC1 antibody (red), and counterstained with the nuclear stain DAPI (blue) and visualized by confocal microscopy at 10× magnification (n = 3). Syncytial formation in RSV infected spheroids at day 3 post-infection was also shown in insets at 40× magnification. (3C) Mucin accumulation and (3D) RSV-F dissemination were measured by the Zeiss LSM710 laser scanning confocal microscope. Mucin intensity fold change was evaluated between infected spheroids and their time matching controls (n = 3). RSV-F intensity fold change was evaluated between day 3 and day 7 infected spheroids (n = 3). Error bars represent ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After 3 and 7 days post-inoculation, spheroids were fixed with 4% paraformaldehyde (416785000 ACROS) for 15 min and permeabilized with 0.2% Triton X-100 (T9284-100 ml Sigma) in PBS for 20 min. Spheroids were blocked with blocking solution [1% BSA (A3912-100 g Sigma) and 0.1% Triton X-100 in PBS] for 1 h followed by overnight incubation with a mixture of mouse anti-RSV fusion protein antibody (MAB8599-Merck Millipore) at 1:200 and rabbit anti-human MUC1 antibody (12123-T24 Sino Biological) at 1:2500 in blocking buffer.

Techniques: Infection, Incubation, Staining, Confocal Microscopy, Microscopy

Journal: bioRxiv

Article Title: Identification of the Metaphyseal Skeletal Stem Cell

doi: 10.1101/2022.09.08.506930

Figure Lengend Snippet: (A, B) Representative multicolor immunofluorescent staining shows the expression of murine SSC panel in P8 femur section. White framed areas are shown in (B) at higher magnifications, which demonstrate CD200 + CD51 + Thy1 − Ly51 − CD105 − CD45 − Ter119 − CD31 − mSSCs at the metaphyseal trabeculae, the periosteum and perichondrium, the secondary ossification center, the articular surface, and the cortical endosteum (white arrows). The yellow arrows pinpoint a cluster of SSCs at the upper metaphyseal region right beneath the hypertrophic zone (HZ) (framed by dashed lines). Scale bar, 500 μm. (C) ScRNA-seq of 5,861 skeletal cells from P7.5 hindlimb bones is visualized by uniform manifold approximation and projection (UMAP) (also see Methods and ). They are unbiasedly clustered into 14 clusters that include bMSCs (cluster 1–3), OLCs (cluster 4–6), periosteal cells (cluster 7), pericytes (cluster 8), and chondrocytes (cluster 9–14). (D) Dot plot shows the expression level of selected cluster-enriched genes in 14 clusters. Dot size indicates the cell fraction of each cluster that expresses listed genes. Purplish grey color intensity indicates scaled average expression level. Black-coded frames indicate murine SSC panel genes Cd200 (CD200), Itgav (CD51), Eng (CD105), Enpep (Ly51), and Thy1. (E) Enriched gene signatures of cluster 1 (mpSSC). Genes with count >1 are identified as positive expressed genes (+); count ≤0 are identified as negative (–). Cells filtered by combined gene expression patterns are labeled with magenta dots and are superimposed on the UMAP plot. (F) Representative multicolor immunofluorescent staining shows the expression of mpSSC markers in P7.5 femur section. White framed areas are shown adjacently at higher magnifications. The upper metaphyseal region right beneath the hypertrophic zone (HZ) is framed by dashed lines. Yellow arrows indicated mpSSCs that are labeled by Sstr2 + or SSC;Pdgfrb + . Scale bar, 500 μm.

Article Snippet: Multiplex antibody panels applied in this study include: GFP (CST, 2956, 1:400), RFP (Rockland, 600-401-379, 1:800), Pdgfrb (CST, 3169, 1:400), Emcn (ThermoFisher, 14-5851-82, 1:800), Sstr2 (Abcam, ab134152, 1:800), N-cadherin (Abcam, ab76011, 1:500), Pdpn (Sino biological, 50256-R066, 1:1000), CD200 (Novusbio, BAF3355, 1:400), CD51 (Abcam, ab208012, 1:400), Ly51 (Sino biological, 50082-T24, 1:1000), Thy1 (Sino biological, 50461-T44, 1:1000), CD105 (Abcam, ab221675, 1:400), CD31 (CST, 77699, 1:400), CD45 (CST, 70257, 1:400), Ter119 (Biolegend, 116201, 1:1000), Ki67 (CST, 12202, 1:400), Lepr (Novusbio, BAF497, 1:200), Hgs (Abcam, ab155539, 1:300), Txnip (Abcam, ab188865, 1:500), F13a1 (Abcam, ab76105, 1:500), Pla2g10 (Abcam, ab166634, 1:800), and Sox9 (Abcam, ab185966, 1:400).

Techniques: Staining, Expressing, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Lon Protease Is Important for Growth under Stressful Conditions and Pathogenicity of the Phytopathogen, Bacterium Dickeya solani

doi: 10.3390/ijms21103687

Figure Lengend Snippet: Confirmation of successful deletion of the lon gene in D. solani IPO 2222: ( A ) PCR analysis of genomic DNA isolated from the D. solani WT (wild type) and Δ lon mutant. The lon gene was replaced by a 1000 bp smaller kanamycin resistance gene. Δlon1 and Δlon2 denote two independent clones ( B ) immunodetection using the anti-Lon E. coli primary antibodies. ( C ) The qPCR analysis with the use of the lon gene-specific primers revealed that no cDNA amplification product was created within 50 cycles.

Article Snippet: Briefly, the Lon protein was detected with the anti- Escherichia coli Lon rabbit antibodies (#40219-T24, Sino Biological Inc., Beijing, China) at dilution 1:2000 followed by incubation with HRP conjugated secondary anti-rabbit antibodies (#31462 Thermo Fisher) diluted 1:50,000.

Techniques: Isolation, Mutagenesis, Clone Assay, Immunodetection, Amplification

Journal: International Journal of Molecular Sciences

Article Title: Lon Protease Is Important for Growth under Stressful Conditions and Pathogenicity of the Phytopathogen, Bacterium Dickeya solani

doi: 10.3390/ijms21103687

Figure Lengend Snippet: Bacterial strains and plasmids used in this study.

Article Snippet: Briefly, the Lon protein was detected with the anti- Escherichia coli Lon rabbit antibodies (#40219-T24, Sino Biological Inc., Beijing, China) at dilution 1:2000 followed by incubation with HRP conjugated secondary anti-rabbit antibodies (#31462 Thermo Fisher) diluted 1:50,000.

Techniques: