Journal: Circulation

Article Title: Wnt5a-mediated neutrophil recruitment has an obligatory role in pressure overload-induced cardiac dysfunction

doi: 10.1161/CIRCULATIONAHA.118.038820

Figure Lengend Snippet: a. Flow cytometry analysis of cardiac tissue from Myelo-KO (Wnt5afl/fl, LysMCre/+) and littermate control (Wnt5afl/fl, LysM+/+) mice before and after TAC at indicated time points. Cells were defined as in Supplemental Figure 1. b. The absolute number of cardiac myeloid cells for each experimental group before and after TAC at the indicated time points (n=4–5 in control and n=3–5 in Myelo-KO group). Numbers are normalized by tissue weight analyzed. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison tests. c. Analysis of transcript expression in the myocardium obtained from Myelo-KO and littermate control mice at 3 days after TAC surgery. 36b4 was used as a reference for normalization (n=7 in control and n=8 in Myelo-KO group). Statistical analysis was performed using Mann-Whitney U test (Il-1b) and unpaired (two-tailed) Student’s t test with Welch’s correction (Il6, Cxcl1, Cxcl2, Cxcl5 and Ccl2). **p<0.01, ***p<0.001, ****p<0.0001. Myelo-KO= myeloid-specific wnt5a knockout mice.

Article Snippet: Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison tests. c. Sequential echocardiographic analysis of Myelo-TG and littermate control mice after TAC at indicated time points (n=10 in each group).

Techniques: Flow Cytometry, Control, Comparison, Expressing, MANN-WHITNEY, Two Tailed Test, Knock-Out

Journal: Circulation

Article Title: Wnt5a-mediated neutrophil recruitment has an obligatory role in pressure overload-induced cardiac dysfunction

doi: 10.1161/CIRCULATIONAHA.118.038820

Figure Lengend Snippet: a. Representative images of heart sections from Myelo-KO (Wnt5afl/fl, LysMCre/+) and littermate control (Wnt5afl/fl, LysM+/+) mice before and after TAC. Samples were collected as indicated time periods. b. Heart weight or lung weight relative tibia length for the experimental groups (n=5–12 in each group). Scale bar indicates 1 mm. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison tests. c. Sequential echocardiographic analysis of Myelo-KO and littermate control mice after TAC at the indicated time points (n=12 in each group). Statistical analysis was performed using two-way repeated measures ANOVA with Sidak’s multiple comparison tests. d. Representative image and tabulation of myocyte cross-sectional analysis based upon wheat germ agglutinin staining of the heart sections from each group at the end of study (n=4 in sham and n=6 in TAC groups). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparison tests. e. Representative data and analysis of Picrosirius red staining of fibrosis in the heart sections from each experimental group of mice (n=4 in sham and n=6 in TAC groups). Scale bar indicates 100 μm. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparison tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Myelo-KO= myeloid-specific wnt5a knockout mice, HW= heart weight, LW= lung weight, TL= tibia length, LVPWTd= left ventricle posterior wall thickness at diastole, LVDd= left ventricle diameter at end-diastole, LVDs= left ventricle diameter at end-systole, FS= fractional shortening, TAC= transverse aortic constriction, CSA= cross sectional area of myocyte.

Article Snippet: Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison tests. c. Sequential echocardiographic analysis of Myelo-TG and littermate control mice after TAC at indicated time points (n=10 in each group).

Techniques: Control, Comparison, Staining, Knock-Out

Journal: Circulation

Article Title: Wnt5a-mediated neutrophil recruitment has an obligatory role in pressure overload-induced cardiac dysfunction

doi: 10.1161/CIRCULATIONAHA.118.038820

Figure Lengend Snippet: a. Representative images of hematoxylin-eosin staining of heart sections from Myelo-TG (Rosa-Wnt5a, LysMCre/+) and littermate control (LysMCre/+) mice after TAC. Samples were collected as indicated time points. Scale bar indicates 1 mm. b. Heart or lung weights were adjusted by tibia length. (n=5–10 in each group). Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison tests. c. Sequential echocardiographic analysis of Myelo-TG and littermate control mice after TAC at indicated time points (n=10 in each group). Statistical analysis was performed using two-way repeated measures ANOVA with Sidak’s multiple comparison tests. d. Myocyte cross-sectional area was evaluated from the analysis of wheat germ agglutinin staining of the heart sections from each group at the end of study (n=4 in sham and n=6 in TAC groups). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparison tests. e. Representative data images and analysis of Picrosirius red staining of heart sections from each group of mice to assess fibrosis (n=4 in each group). Scale bar indicates 100 μm. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparison tests. *p<0.05, **p<0.05, ***p<0.001, ****p<0.0001. Myelo-TG= myeloid-specific wnt5a overexpression mice, HW= heart weight, LW= lung weight, TL= tibia length, LVPWTd= left ventricle posterior wall thickness at diastole, LVDd= left ventricle diameter at diastole, LVDs= left ventricle diameter at systole, FS= fractional shortening, CSA= cross sectional area of myocyte.

Article Snippet: Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison tests. c. Sequential echocardiographic analysis of Myelo-TG and littermate control mice after TAC at indicated time points (n=10 in each group).

Techniques: Staining, Control, Comparison, Over Expression

Journal: Biomaterials

Article Title: Generation of human tonsil epithelial organoids as an ex vivo model for SARS-CoV-2 infection

doi: 10.1016/j.biomaterials.2022.121460

Figure Lengend Snippet: Innate immune response and SARS-CoV-2 (co)receptor expression in tonsil epithelial organoids. (A) Human cytokine array for secreted proteins from the tonsil organoids before (−) and after (+) LPS treatment at a concentration of 10 μg/mL for 24 h. (B) Changes in cytokine secretion levels were determined by densitometric analysis of the blots shown in (A). Values relative to the control levels (- LPS) are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by two-way ANOVA with Sidak's pairwise multiple comparison test. ***, p < 0.001; ****, p < 0.0001. n.d., not detected. A.U., arbitrary units. (C) Effect of LPS-stimulated culture supernatants on cell migration. HL-60 cells were treated with mock- or tonsil organoid culture medium before (- LPS) and after LPS stimulation (+LPS). Numbers of migrated cells are presented as means ± SEM of the three independent experiments. Two-way ANOVA with Sidak's multiple comparison test was used for statistical analysis. n.s., not significant. *, p < 0.05. (D) Immunofluorescence staining of ACE2 (left), TMPRSS2 (middle) and furin (right) (red) expressed in tonsil organoids on day 15 as well as in tonsil tissues. NGFR and MUC1 (green) are stained as epithelial markers for the basal and suprabasal/superficial layers, respectively. Merged images with Hoechst 33342 staining (blue) are presented on the right side of each panel. Zoom images of white rectangles are displayed at the bottom. Scale bars are 100 μm. (E) Quantitative RT-PCR to detect ACE2 (left), TMPRSS2 (middle) and Furin (right) mRNA transcripts on days 5, 10 and 15 in tonsil organoids originating from three different donors. Statistical analysis was performed by ordinary one-way ANOVA with Tukey's multiple comparison test. n.s., not significant. *, p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparison test. n.s., statistically not significant. (E) Organoid samples from three different donors (21–26, 21–27 and 22–33) were infected with SARS-CoV-2 (MOI, 0.1) and treated with increasing concentrations of remdesivir.

Techniques: Expressing, Concentration Assay, Migration, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Biomaterials

Article Title: Generation of human tonsil epithelial organoids as an ex vivo model for SARS-CoV-2 infection

doi: 10.1016/j.biomaterials.2022.121460

Figure Lengend Snippet: Infection and amplification of SARS-CoV-2 in human tonsil epithelial organoids. (A, B) Immunofluorescence staining for viral spike protein (A) and dsRNA (B) (green) and a cellular receptor, ACE2 (red), in tonsil epithelium organoids on day 3 after SARS-CoV-2 infection at MOI of 0 (Mock), 0.1, or 1. Scale bar is 100 μm. Merged images with nuclei (blue) and their zooms are presented in the right panels. (C) Quantitative RT-PCR for detecting viral RNA in the culture supernatants of tonsil organoids infected with SARS-CoV-2 at an MOI of 0.1 or 1 at various time points after infection. (D) Determination of the amount of infectious viral particle in culture supernatants of SARS-CoV-2–infected tonsil organoids from the same donor of (C). Viral titers were measured by infection of fresh Vero CCL-81 cells with organoid culture medium for 2 days and by staining with anti-spike protein antibody, as described in Materials and Methods . ( E, F ) Both viral RNA copies (E) and infectious SARS-CoV-2 titers (F) in culture supernatants were determined independently from three additional tonsil organoids (donors 20-13, 20-14 and 2016). Two-way ANOVA with Dunnett's multiple comparison test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (G) TEM analysis showing accumulation of SARS-CoV-2 particles on the apical surface of tonsil organoids on day 3 (lower) but not on 3 h (upper) after infection at an MOI of 0.1. Specific in the red rectangle are enlarged at right to show budding-out of viral particles on 72 h post-infection. Mock-infected samples are used as a control for showing pathogen-free samples. Scale bars are 1 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparison test. n.s., statistically not significant. (E) Organoid samples from three different donors (21–26, 21–27 and 22–33) were infected with SARS-CoV-2 (MOI, 0.1) and treated with increasing concentrations of remdesivir.

Techniques: Infection, Amplification, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Biomaterials

Article Title: Generation of human tonsil epithelial organoids as an ex vivo model for SARS-CoV-2 infection

doi: 10.1016/j.biomaterials.2022.121460

Figure Lengend Snippet: Gene expression changes in human tonsil epithelial organoids in response to SARS-CoV-2 infection. (A) PCA plot of time-course microarray samples from three different donors, 21-3 (black), 21-4 (red) and 20–23 (blue). PCA scores on PC1 (63%) and PC2 (6%) are grouped on the basis of time course, 0 (Mock), 3, 24 and 72 h post-infection. (B) Heatmap showing 395 genes upregulated after SARS-CoV-2 infection in tonsil epithelial organoids from three different donors, 21-3 (simply 3), 21-4 (4), and 20–23 (23). Red and blue represent upregulation and downregulation, respectively (with > 2-fold changes). (C) Top 10 enriched GO categories of differentially upregulated genes in SARS-CoV-2-infected samples. (D) Representative upregulated gene clusters colored in (B) are enlarged with their gene lists. (E) Quantitative RT-PCR of upregulated genes, ANXA9 , LOR , ATP6V1C2 and SPTLC3 , which are randomly selected among DEGs, marked with red spots in (D). Values are presented as means ± SEM of three independent experiments from the three donors. (F) Heatmap showing 777 genes downregulated after SARS-CoV-2 infection in tonsil epithelial organoids from three different donors, 21-3 (3), 21-4 (4), and 20–23 (23). (G) Top 10 enriched GO categories of differentially downregulated gene in SARS-CoV-2-infected samples. (H) Representative downregulated gene clusters colored in (F) are enlarged with their gene lists. (I) Quantitative RT-PCR of downregulated genes, ATF3 , JUN , RSG2 and HSPA6 , which are randomly selected among DEGs, marked with blue spots in (D). HSPA6 is downregulated in the IL-5 regulation pathway, thus not marked with a dot. Values are presented as means ± SEM of three independent experiments from the three donors. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparison test. n.s., statistically not significant. (E) Organoid samples from three different donors (21–26, 21–27 and 22–33) were infected with SARS-CoV-2 (MOI, 0.1) and treated with increasing concentrations of remdesivir.

Techniques: Expressing, Infection, Microarray, Quantitative RT-PCR

Journal: Biomaterials

Article Title: Generation of human tonsil epithelial organoids as an ex vivo model for SARS-CoV-2 infection

doi: 10.1016/j.biomaterials.2022.121460

Figure Lengend Snippet: Evaluation of antiviral activity of remdesivir in SARS-CoV-2-infected tonsil organoids. (A) Quantitative RT-PCR showing inhibition of viral RNA titers on day 2 after treatment of SARS-CoV-2–infected organoids with increasing concentrations of remdesivir (0.1, 1, or 10 μM). Mock: non-infected control. RNA copies are presented as means ± SEM of four independent experiments using tonsil organoids from a single donor (no. 20-16). Statistical analysis was performed by ordinary one-way ANOVA with Dunnett's multiple comparison test. **, p < 0.01. (B) Western blot analysis showing reduction of viral spike and nucleocapsid proteins in two different organoids samples (donors 20-16 and 21–36, respectively) in the presence of remdesivir. Beta-actin was used as a loading control. (C) Reduction of nucleocapsid protein by remdesivir was quantified from Western blot analysis with three different organoid samples (donors 21-16, 21–34 and 21–36). Statistical analysis was performed by two-way ANOVA with Dunnett's multiple comparison test. ****, p < 0.0001. (D) Cell viability test. Organoids from three different donors (21–26, 21–27 and 22–33) were mock infected or infected with SARS-CoV-2 alone (MOI, 0.1) or in the presence of 100 μM remdesivir. On day 2, organoids from three different wells were dissociated with trypsin-EDTA and individually stained with trypan blue. Live and dead cells were counted by using an automated microscopic cell counter. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparison test. n.s., statistically not significant. (E) Organoid samples from three different donors (21–26, 21–27 and 22–33) were infected with SARS-CoV-2 (MOI, 0.1) and treated with increasing concentrations of remdesivir. On day 2 post-infection, RNA samples were harvested from culture supernatants for qRT-PCR against the viral nucleocapsid gene. EC 50 values are calculated in three independent experiments and expressed as mean ± SD. (F) Confocal miscopy showing reduction of viral spike protein by remdesivir. Tonsil epithelial organoids (donor 21–36) were mock-infected (upper) or infected with SARS-CoV-2 at an MOI of 0.1. The infected organoids were treated with DMSO (middle) or remdesivir (10 μM; lower) for 2 days. Viral spike protein (blue) and its binding receptor ACE2 (red) were stained with their specific antibodies. Nuclei (blue) were counter-stained with Hoechst 33342. Merged images and their zooms are displayed on the right side. Scale bar is 100 μm.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparison test. n.s., statistically not significant. (E) Organoid samples from three different donors (21–26, 21–27 and 22–33) were infected with SARS-CoV-2 (MOI, 0.1) and treated with increasing concentrations of remdesivir.

Techniques: Activity Assay, Infection, Quantitative RT-PCR, Inhibition, Western Blot, Staining, Binding Assay