Fig 1: Bone-marrow mononuclear cell characterization before and after specific Dynabead depletions. Percentage of CD45-, CD34-, CD3-, CD19-, CD11b-, CD31-, and Sca-1-positive cells evaluated by flow cytometry analysis of bone-marrow derived mononuclear cells before and after magnetic depletions. Representative depletions are shown. Data are presented as mean + SD (n = 3). ∗, significance compared with BMDMC before depletion.
Fig 2: Study design. (A): Protocol of allergic airway inflammation induction. OP administration of PBS (naïve) or AHE solution on days 0 and 7 to initiate the immune response, then challenged for 3 days on days 14, 15, and16 with OP inoculations of AHE. Cell administration was performed on day 14; harvest was on day 19. (B): Experimental groups. Animals exposed to AHE solution (A) were randomly divided to receive treatment with the vehicle PBS, HLFs, mMSCs, BMDMCs, and BMDMCs depleted of CD45, CD34, CD11b, CD3, CD19, CD31, and Sca-1 positive cells. Abbreviations: #, oropharyngeal Aspergillus hyphal extract solution administered; ↑, human lung fibroblasts, mouse mesenchymal stem/stromal cells, bone marrow-derived mononuclear cells and depleted bone marrow-derived mononuclear cells administered; A, animals exposed to AHE solution; AHE, Aspergillus hyphal extract; BMDMC, bone marrow-derived mononuclear cell; HLF, human lung fibroblast; mMSC, mouse mesenchymal stem/stromal cell; OP, oropharyngeal; P, group receiving phosphate-buffered saline; PBS, phosphate-buffered saline.
Fig 3: The NLRP3 inflammasome is induced in Cdx mutants.A–G, immunoblot analyses for (A and B) NLRP3; (C–E) caspase-1; and (F and G) IL-1β. The lower molecular weight bands for the latter two immunoblots represent the processed forms of the proteins. H–J, flow cytometric dot plots of freshly isolated primary intestinal cells for Ly6C- and Ly6G-positive cells (indicated by circles in H and I) obtained from WT (H) and DKO (I) colon and quantified in (J). Living cells were selected based on the absence of Zombie yellow staining. Cell sorting was based on the expression of either EpCAM or CD45 in the double-stained samples. Cells were gated based on size and granularity using FSC-A versus SSC-A to eliminate debris and clumped cells. Error bars represent the standard deviation from the mean of three independent biological experiments. Statistical significance was calculated using two-tailed t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. DKO, double KO; FSC-A, forward scatter area; IL-1β, interleukin 1β; NLRP3, NLR family, pyrin domain containing 3; SSC-A, side scatter area.
Fig 4: Differential regulation of the BM niche by >52w and 3w AML. a Schematic overview of RNA seq data acquisition and analysis. b Plot of principal component analysis of gene expression in NH9FL, NH93w, NH910w and NH9>52w myeloid leukaemias, n = 3 in each group. c Flow cytometric gating strategy used to identify endothelial cells (EC), mesenchymal cells (MSC) and osteoblasts (OB) in the stroma (CD45−Ter119−). Leukaemic burden assessed by d %GFP in BM and e WBC count. Analysis of percentage ECs (top) and MSCs (bottom) in the stroma of central marrow of mice transplanted with f NH9+ AML blasts or g healthy LSKs of foetal (LSKFL) or >52w (LSK>52W) origin. Analysis of percentage OBs (bottom) and MSC (top) in the stroma of the compact bone of mice transplanted with h NH9+ AML blasts or i LSKFL or LSK>52W. d–i Graphs depict individual values with mean ± s.d. of mice transplanted with leukaemic blasts from NH93W (n = 13) or NH9>52W (n = 9) (only mice with >30% GFP in the bone marrow are included, and individual mice are indicated with different symbols on the graphs), and LSKFL(n = 4) or LSK>52w(n = 4) from healthy mice. NT not transplanted (n = 4). Significance determined by Student’s t-test, *p < 0.05, **p < 0.01
Fig 5: Knockout of RAB6 promoted the self-renewal and proliferation of AEC2s.a The enriched AEC2 cell population was isolated by selection of the CD24− SFTPC+ subset from epithelial cell populations (EpCAM+CD31−CD34−CD45−) in lung tissues by FACS. b The localization and expression of RAB6 and SFTPC proteins in WT and RAB6−/− AEC2 cells were detected by immunofluorescence (Green, RAB6; red, SFTPC; blue, DAPI) (Representative immunofluorescence images are shown, n = 3). c WT and RAB6−/− AEC2 cells were exposed to PM2.5 (100 μg/ml) or saline for 48 h. The apoptosis of AEC2 cells was determined by FACS (Representative FACS images are shown, n = 3). d Statistical analysis of apoptosis rate. e The colony forming efficiency (CFE) of WT and RAB6−/− AEC2 cells was examined by plate colony formation assay (Representative images are shown, n = 3). f Statistical analysis of CFE (represented by colony numbers). g Self-renewal (spherication ability) of WT and RAB6−/− AEC2 cells exposed to PM2.5 or saline was detected by 3D culture (Representative images are shown, n = 3). h–j The relative RNA expression of SOX2 (h), OCT4 (i), and NANOG (j) was measured by qRT-PCR. (n = 3; unpaired two-tailed t test. **P < 0.01; ***P < 0.001. Bar graphs represent the mean ± SEM for d, f, h–j).
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