Fig 1: 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 2: Increased mast cell maturation and degranulation due to TAZ deficiency. BMMCs were established from the bone marrow of WT and TAZ KO mice and cultured in the presence of IL-3 for 35 days. A Flow cytometry analysis of BMMCs expressing FcεR1 and c-kit. Data are expressed as the mean ± SEM (n = 5). B Immunofluorescence staining for TAZ and LAMP1 in WT and TAZ KO BMMCs. DAPI was used for nuclear staining. C Giemsa staining of BMMCs upon C48/80 stimulation. D The β-hexosaminidase release assay in WT and TAZ KO BMMCs after treatment with C48/80. Data are expressed as the mean ± SEM (n = 8). ***, p < 0.0005 by ANOVA with post-hoc Tukey′s HSD test. E BMMCs were stained with antibodies against TAZ, LAMP1, and HRF and subsequently incubated with DAPI, followed by observation with confocal fluorescence microscopy. F Culture supernatants of BMMCs stimulated with LPS for 24 h were subjected to HRF ELISA. G Total RNA was prepared from LPS-stimulated BMMCs and subjected to determine the relative transcript levels of TNF-α, IL-1β, and IL-13
Fig 3: Deep mutational scanning of the EGFR L858R kinase domain in Ba/F3 cells.A Map of pHAGE-EGFR-L858R with kinase domain annotated. B Frequency of mutations count per cDNA. C Per-codon frequency of the indicated mutation types. D Cumulative distribution of read counts for all expected variants in the library. E Density plot of plasmid library codon counts. F Enrichment of library transduced Ba/F3 cells after IL-3 withdrawal. G Positive selection screening scheme.
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