Journal: Biochimica et Biophysica Acta. Molecular Basis of Disease

Article Title: Single cell RNA-seq resolution revealed CCR1 + /SELL + /XAF + CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19

doi: 10.1016/j.bbadis.2023.166707

Figure Lengend Snippet: The differential activation of CD14 and CD16 monocytes responding to endothelial and epithelial dysfunction. The profile scores of molecules mediating the interplays of classic monocytes with vascular endothelial cells (A-B) and non-classic monocytes with alveolar epithelial cells (C—D). E. Immunostaining for CDH5 and γH2AX in coronary artery cryo-section between KD patient and healthy donor, indicating endothelial injuries in KD. F. Expression level of CCR1 , DYSF , SELL , LMNB1, and XAF1 in CD14 classical monocytes among five groups. G. UMAP projection of CCR1 , DYSF , SELL , LMNB1, and XAF1 positive cells, respectively . H. UMAP projection of CCR1 , SELL and XAF1 triple positive monocytes among five groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Wilcoxon rank-sum test, adjusted for Bonferroni post hoc test, had been applied.

Article Snippet: Cell smears fixated in 4 % PFA were blocked in blocking buffer (PBS containing 1 % BSA, 0.1 % Triton X-100, 5 % goat serum) for 1 h at room temperature, followed by incubation with rabbit anti-Dysferlin (1:200, Abcam, cat # ab124684), rabbit anti-XAF1 (1:200, Abcam cat #ab2254) antibodies and mouse anti-CD14 (1:200, Abcam cat #ab181470) diluted in PBS containing 1 % BSA, 5 % goat serum at 4 °C overnight.

Techniques: Activation Assay, Immunostaining, Expressing

Journal: Biochimica et Biophysica Acta. Molecular Basis of Disease

Article Title: Single cell RNA-seq resolution revealed CCR1 + /SELL + /XAF + CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19

doi: 10.1016/j.bbadis.2023.166707

Figure Lengend Snippet: SELL+/CCR1+/XAF1+ CD14 monocytes enhanced the adhesion and damages to endothelial cells. A. Flow cytometry identified the higher percentages and median fluorescence intensity of SELL, CCR1, and LMNB1 in KD and COV. B. Immunostaining for CD14 and DYSF, XAF1 in isolated PBMCs. C. The ratio of DYSF positive CD14 monocytes in total classic monocytes among KD, COV, FLU, and healthy donors. And the median fluorescence intensity of XAF1 in CD14 monocytes among KD, FLU, and healthy donors. D-E. THP-1 had been stained with Calcein AM and transfected siRNAs of CCR1 , DYSF , SELL , LMNB1 , and XAF1 before co-culture with HUVECs. Then the numbers of adhesion THP-1 with HUVECs were counted by every 20× field view. F. The expressions of TNFa and IL6 in HUVECs, and the ratio of γH2AX + cells in HUVECs after co-cultured with THP-1, which had been transfected with siRNAs of CCR1 , DYSF , SELL , LMNB1 , and XAF1 . G. The inhibition of SELL , CCR1 and XAF1 together in THP-1 significantly reduced the adhesion between monocytes and endothelial cells. H. Collaborated inhibiton of SELL , CCR1 and XAF1 in THP-1 decreased the expression of IL6 and TNF-α in HUVECs with lower ratio of rH2AX+ cells after co-culture with THP-1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Two-way analysis of variance with Bonferroni post hoc test was performed to analyze data. Bar, 100 μm.

Article Snippet: Cell smears fixated in 4 % PFA were blocked in blocking buffer (PBS containing 1 % BSA, 0.1 % Triton X-100, 5 % goat serum) for 1 h at room temperature, followed by incubation with rabbit anti-Dysferlin (1:200, Abcam, cat # ab124684), rabbit anti-XAF1 (1:200, Abcam cat #ab2254) antibodies and mouse anti-CD14 (1:200, Abcam cat #ab181470) diluted in PBS containing 1 % BSA, 5 % goat serum at 4 °C overnight.

Techniques: Flow Cytometry, Fluorescence, Immunostaining, Isolation, Staining, Transfection, Co-Culture Assay, Cell Culture, Inhibition, Expressing

Journal: PLoS ONE

Article Title: Novel PDE4 Inhibitors Derived from Chinese Medicine Forsythia

doi: 10.1371/journal.pone.0115937

Figure Lengend Snippet: A. 5×10 5 RAW264.7 cells were seeded in 96 wells for 18 h. Cells were primed with compounds at different concentration for 3 h before treated with LPS (1 ng/ml) for additional 8 h. TNF cytokine releases were monitored by ELISA. B. PBMC (0.2 ml at 1×10 5 /ml) were primed with compounds at different concentration for 3 h before treated with LPS (1 ng/ml) for additional 8 h. TNF cytokine releases were monitored by ELISA. % of TNF secretion were calculated and graphed. C. Summary of compound IC 50 . The data represent n = 3–6 experiments.

Article Snippet: IL1β, TNFα, IL6 mouse ELISA kit, human TNFα were from R&D systems.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay