Journal: Nature Communications

Article Title: Distinct fibroblast subsets regulate lacteal integrity through YAP/TAZ-induced VEGF-C in intestinal villi

doi: 10.1038/s41467-020-17886-y

Figure Lengend Snippet: a Diagram depicting the generation of tdTomato rPβC mouse and PDGFRβ + cell-specific expression of tdTomato from 8-week old and their analyses at 10-week old. b Illustration of droplet-based single-cell RNA sequencing (scRNA-seq) of PDGFRβ + tdTomato + IntSCs sorted from the small intestine of tdTomato rPβC mice. c Visualization of unsupervised clustering of 7 distinct PDGFRβ + IntSC clusters by Uniform manifold approximation and projection (UMAP) in the small intestine of tdTomato rPβC mice. vFB, intestinal villi fibroblast; FB, fibroblast; SMC, smooth muscle cell; MC, mural cell. d Heatmap displaying the scaled expression patterns of top ten differentially expressed genes for random sampled cells (maximum thousand cells) for each indicated clusters. e List of representative marker genes of each of the seven PDGFRβ + IntSC clusters (left) and violin plots (right) showing the expression of top-ranking marker genes for each cluster. Log normalized read counts as y -axis (normalized expression). f UMAP visualization of unsupervised clustering of seven distinct PDGFRβ + IntSC clusters in the small intestine of Lats1/2 iΔ - tdTomato rPβC mice. g Gene expression levels of Vegfc and Vegfa in tdTomato rPβC and Lats1/2 iΔ - tdTomato rPβC mice projected on UMAP plot. Note that specific subsets of PDGFRβ + IntSCs (vFB1-3) in the small intestine of Lats1/2 iΔ - tdTomato rPβC mice show higher expression of Vegfc compared with those of tdTomato rPβC mice. h , i Representative images of PDGFRβ + IntSCs in WT mouse reveal expressions of each fibroblast-specific markers: PAI-1 + vFB1, Serpina3n + vFB2, P2X1 + vFB3, Ackr4 + or Grem1 + FB4, and PDGFRα + Sox6 + FB5. Each white box in the lower left corner is a magnified view. White asterisks indicate lacteals and white arrowheads indicate each fibroblast cluster-specific cell type stained with the indicated marker. Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 20 μm. j Schematic images depicting the anatomic distribution of indicated markers for vFB1-3, FB4, FB5, SMC, MC, capillary plexus, and lacteal in intestinal villi of adult WT mouse. vFB1-3 are uniformly distributed around the lacteal, whereas FB4 is mainly located in the submucosal area and FB5 is mostly placed under the intestinal epithelium. Black dashed boxes are magnified in the right panels.

Article Snippet: The following primary and secondary antibodies were used in the immunostaining with smFISH: anti-PDGFRβ (rat monoclonal, ab91066, Abcam); anti-Shisa3 (rabbit polyclonal, TA320118, Origene); anti-P2X1 (rabbit polyclonal, APR-001, Alomone labs) and Alexa Fluor 488-, Alexa Fluor 594-, Alexa Fluor 647-conjugated secondary antibodies were purchased from Jackson ImmunoResearch.

Techniques: Expressing, RNA Sequencing Assay, Marker, Staining

Journal: Nature Communications

Article Title: Distinct fibroblast subsets regulate lacteal integrity through YAP/TAZ-induced VEGF-C in intestinal villi

doi: 10.1038/s41467-020-17886-y

Figure Lengend Snippet: a Representative images of vFB3 (P2X1 + Serpina3n + PDGFRβ + ) and vFB1-2 (P2X1 - Serpina3n + PDGFRβ + ) in WT mouse. Similar findings were observed in n = 4 mice from two independent experiments. White and yellow dotted boxes are magnified in the upper right and lower right panels, respectively. Red and yellow arrowheads indicate vFB3 and vFB1-2, respectively. Scale bar, 20 μm. b Representative images of vFB1 (Serpina3n + Fosb + Shisa3 + PDGFRβ + ) and vFB2 (Serpina3n + Fosb - Shisa3 + PDGFRβ + ) in WT mouse. Similar findings were observed in n = 4 mice from two independent experiments. White and yellow dotted boxes are magnified in the upper right and lower right panels, respectively, for each image. White and yellow arrowheads indicate vFB1 and vFB2, respectively. Scale bars, 20 μm. c Representative images of Vegfc single-molecule fluorescence in situ hybridization (smFISH) in intestinal villi of tdTomato rPβC and Lats1/2 iΔ - tdTomato rPβC mice. Similar findings were observed in n = 4 mice/group from two independent experiments. White dotted boxes are magnified in the right panel. Scale bars, 20 μm. d Representative images of Vegfc smFISH in vFB1-3 of WT and Lats1/2 iΔ - tdTomato rPβC mice. Similar findings were observed in n = 4 mice/group from two independent experiments. White and yellow dotted boxes are magnified in the upper right and lower right panels, respectively, for each image. Scale bars, 20 μm.

Article Snippet: The following primary and secondary antibodies were used in the immunostaining with smFISH: anti-PDGFRβ (rat monoclonal, ab91066, Abcam); anti-Shisa3 (rabbit polyclonal, TA320118, Origene); anti-P2X1 (rabbit polyclonal, APR-001, Alomone labs) and Alexa Fluor 488-, Alexa Fluor 594-, Alexa Fluor 647-conjugated secondary antibodies were purchased from Jackson ImmunoResearch.

Techniques: Fluorescence, In Situ Hybridization

Journal: Scientific Reports

Article Title: Dysfunction of low-density neutrophils in peripheral circulation in patients with sepsis

doi: 10.1038/s41598-021-04682-x

Figure Lengend Snippet: Reduced chemotactic function of LDNs. ( A ) Neutrophil chemotaxis toward fMLP was assayed. CD (chemotactic distance, μm), CCR (chemo cell ratio, cell number of zone I + II + III/Total cells in the side well, %), CI (chemo index, cell number of zone II + III/ Total number of chemotactic cells, %). ( B – D ) The evaluation indicators CD, CCR, and CI were significantly reduced in neutrophil chemotactic function evaluations of LDN. ( E ) The expression of P2X1 on HDNs and LDNs was analyzed using flow cytometry.

Article Snippet: Antibodies used in the experiment include CD66b (G10F5, BD Pharmingen, USA), CD10 (HI10α, Biolegend, USA) CXCR4 (12G5, Biolegend, USA) and P2X1 (APR-022-F, Alomone labs, Israel).

Techniques: Chemotaxis Assay, Expressing, Flow Cytometry