Fig 1: SLex mediated rolling of eosinophils on P-selectin-expressing cells without affecting the migration. (A) The schedule of eosinophil induction. The mouse hematopoietic progenitor cells were isolated from the bone marrow using the MojosortTM hematopoietic progenitor cell isolation kit. FLT3-Ligand and SCF were added to the culture medium on days 0 and 2. The medium was partly exchanged with medium supplemented with IL-5 on days 4, 7, and 10. (B) The harvested eosinophils were analyzed using flow cytometry. The mature eosinophils were defined as CD45+Siglec-F+CD11b+CCR3High. The experiment was independently repeated twice. (C) The rolling assays were performed, and the videos were taken using a CMOS camera equipped on a microscope at the speed of 20 photos per second for 30 s. In total, the stuck of 600 pictures were analyzed using Trackmate in Fiji ImageJ after subtracting the background. The rolling cells are circled with purple, and their rolling tracks are drawn in yellow by the software. The experiments were repeated twice independently. (D) The speed of the rolling cells was calculated using the Trackmate in Fiji ImageJ. In the WT eosinophil group, most of the cells moved between 5 and 15 μm/s. No rolling cells were detected in the DKO eosinophil (DKO Eosinophils) and F2-treated WT eosinophil groups (WT Eosinophils with F2). (E) A migration assay was performed using transwells, and the percentage of migrated eosinophils into the lower chamber was shown. Cells from the murine bone marrow was pretreated with or without F2 mAb and used for the migration assay. The lower chamber contained mCCL11 or medium only. ** p < 0.01. A p-value of less than 0.05 was considered significant.
Fig 2: IL-9 enhances MC progenitor proliferative capacity. (A) WT (CD45.1+) and Il9r−/− (CD45.2+) bone marrow cells were transferred to lethally irradiated Boy/J x C57BL/6J F1 mice and after 3 months to allow repopulation of the immune system, mice were treated with HDM for 6 weeks. Flow cytometry analysis of CD45.1+ and CD45.2+ of lung MCp. (n = 10). (B–C) BMMC from WT mice were cultured for 2 weeks in IL-3 and SCF in RPMI. BMMC were harvested and stimulated with IL-9 (40 ng/ml) for 2 hours to assess intracellular Ki67 using flow cytometry. (B) flow cytometry plots of Ki67 staining in BMMC with WT and Il9r−/− BMMC. (C) Flow cytometry analysis of Ki67 frequencies in MCp and mMC (n = 4). (D) WT mice were intranasally treated with rIL-9 for 3 days. Flow cytometry of Ki67 gMFI was measured from bone marrow and lung MC (n = 5); E, flow cytometry analysis of Ki67 was assessed in lung MC from 6-week HDM-treated WT mice or PBS controls (n = 3). (F–I) Il9−/− and WT mice were treated intranasally with HDM 3x/week for 6 weeks. Ki67 expression was assessed via flow cytometry in (F–G) bone marrow and (H–I) lung MC (n = 3). Each data point represents an individual mouse. Data are representative of two independent experiments with similar results. Error bars indicate ± standard error of mean. Statistical significance was determined by analysis of variance, followed by Sidak’s multiple comparison test (A), Mann-Whitney U test (C–D), and Student’s t test (E–I). CD = clusters of differentiation; gMFI = geometric mean fluorescence intensity; HDM = house dust mite; IL = interleukin; MC = mast cell; MCp = MC progenitors; mMC = mature MC; ns = not significant; PBS = phosphate buffered saline; PE = R-phycoerythrin; r = recombinant; SSC = side scatter; WT, wild type.
Fig 3: High-GATA3 expression induced by G3SE is essential for the development from ILCP to ILC2.a Flow cytometry analysis of BM cells. Frequencies and numbers of CLPs (Lin−CD127+CD135+LPAM-1− cells), PD-1−ST2− (Lin−CD127+CD135−LPAM-1+PD-1−ST2− cells), IL17RB−PD-1+ (Lin−CD127+CD135−LPAM-1+PD-1+ST2−IL17RB− cells), IL17RB+PD-1+ (Lin−CD127+CD135−LPAM-1+PD-1+ST2−IL17RB+ cells), and ST2+ ILC2s (Lin−CD127+CD135−LPAM-1+PD-1−ST2+ cells) were evaluated; n = 6. Lin: CD3ε, CD4, CD5, CD8a, CD11b, CD11c, CD19, NK1.1, Gr1, B220, Ter119. b, c Sorted ILCPs from the BM of WT mice and G3SEKO mice were cultured on OP9-DL1 cells in the presence of IL-7 and SCF for 5 days. Frequencies of Lin−CD25+PD-1− cells, Lin−CD25−PD-1− cells, Lin−CD25−PD-1+ cells (b), and Lin−CD25+GATA3+ cells (c) were evaluated, n = 3. d ILCPs sorted from the BM of WT mice and G3SEKO mice were cultured on OP9-DL1 cells. Cells were infected with GATA3-expressing or empty retrovirus on day 1. The infected cells (Thy1.1+ cells) were analyzed on day 6. WT, G3SEKO+empty-RV: n = 5, G3SEKO+Gata3-RV: n = 4. Each data point indicates one mouse from six (a) or three (b–d) independent experiments. Data are presented as mean ± SE. Statistical analysis was performed using unpaired, two-sided Welch’s t-test. p values are shown on the graphs. Source data are provided as a Source Data file.
Fig 4: In vivo transplantation of SCF-soaked/CM-DiI-labeled beads into the testicular interstitium induces proliferation of differentiating spermatogonia in Cldn11−/− mice.a Immunohistochemistry of testis sections prepared from busulfan-treated Cldn11+/− and Cldn11−/− mice using anti-SCF and anti-ZO1 antibodies. b Schema for in vivo transplantation of SCF-soaked/CM-DiI-labeled beads into the interstitium of Cldn11−/− mouse testes. Appearance of a SCF-soaked/CM-DiI-labeled bead is shown. CM-DiI labels seminiferous tubules in proximity to the beads. c Whole-mount immunofluorescence staining using anti-KIT antibody on seminiferous tubules from Cldn11−/− mouse testes, with bovine serum albumin (BSA)- or SCF-soaked beads transplanted into the interstitium. Seminiferous tubules with or without CM-DiI labeling are shown as CM-DiI+ and CM-DiI-, respectively. White dotted lines outline the seminiferous tubules. d BSA- and SCF-soaked beads were separately transplanted into distinct testes of a Cldn11−/− mouse. Transplantation was performed using six biologically independent Cldn11−/− mice. After, 67 and 72 seminiferous tubules labeled with CM-DiI derived from BSA- or SCF-soaked beads, respectively, were analyzed. The number of KIT+ cells per 100 μm seminiferous tubule labeled with CM-DiI was quantified. Red dots indicate biological replicates of mice. Data are shown as mean ± SD and were analyzed by Welch’s t-test. e Working model showing the role of CLDN11 in maintenance of differentiating spermatogonia. CLDN11-based Sertoli cell tight junctions (SCTJs) physically divide seminiferous tubules into adluminal and basal compartments. KIT-positive differentiating spermatogonia are located at the basal compartment. Interaction of Sertoli cell-derived SCF with KIT is essential for survival of differentiating spermatogonia. SCF accumulates at the basal compartment through Sertoli cell polarization after SCTJ formation, presumably promoting KIT activation in differentiating spermatogonia. Cldn11 knockout delocalizes SCF from the basal compartment and reduces the number of differentiating spermatogonia, likely due to a decrease in KIT activity. Scale bars: 10 μm (a), 100 μm (b), and 50 μm (c).
Supplier Page from BioLegend for Recombinant Mouse SCF (carrier-free)