Fig 1: Germination assay of conidia of A. fumigatus co-cultured with HUVECs and neutrophils. HUVECs were seeded in 24-well plate (3 × 105 cells per well) overnight before the conidia of A. fumigatus were added to the wells at a cell:spore ratio of 1:2. Neutrophils (2 × 105 cells per well) were added 2 h after adding conidia. Representative pictures of conidia were taken at indicated time with WT (A), CSF3– /– (B) or CSF3overexpressing (C) HUVECs and neutrophils. The percentage of germinated conidia was calculated (D). (E–L) Neutrophils (2 × 105 cells per well) were added to a 0.4-µm Transwell chamber (E,G,I,K) or without the Transwell chamber (F,H,J,L) 2 h after adding conidia, and both the neutrophil cells and supernatant were harvested 6 h later. The gene expression of IL1B (E,F), IL8 (G,H), and NOS2 (I,J) were tested by qPCR, and granulocyte-colony-stimulating factor (MPO) was also analyzed (K,L). WT, wild type HUVEC; KO, CSF3 knockout HUVEC (described in Supplementary Figure 2); OV, HUVECs transfected with CSF3 plasmid 48 h before stimulation with conidia. *p < 0.05 compared with WT. Original magnification × 100. Black arrow, germinating conidia.
Fig 2: Combination of G-CSF and rhTPO exerts greater effect on inhibiting leukemia growth than G-CSF or rhTPO alone in vitro.A Volume of xenograft tumors formed in nude mice injected with KG1a cells and treated with combination of G-CSF and rhTPO, G-CSF or rhTPO. B Representative images of xenograft tumors formed in nude mice injected with KG1a cells and treated with combination of G-CSF and rhTPO, G-CSF or rhTPO. The bar graph showed weights of xenograft tumors. *P < 0.05, ***P < 0.001.
Fig 3: ELISA data showing levels of G-CSF in serum and bone marrow in control and mouse model of periodontitis. A significantly greater level of G-CSF in serum from the periodontitis group, compared with the control group (A). The amount of G-CSF in bone marrow tended to be upregulated in the periodontitis group, but there was no difference between the two groups (B). Data are presented as mean ± SD. *P < 0.05 using two-tailed Students t-test. ns, no differences
Fig 4: The G-CSF-mediated JAK2/STAT3 signaling was promoted by SIPS in CHRF, K562 cell and primary cultured BMMNCs.After 24-h incubation with SIPS, the protein expression levels of G-CSF, M-CSF, P-JAK2, P-STAT3 in CHRF cells (a), K562 cells (b) and BMMNCs (c) were analyzed by western blotting. The protein levels were normalized by the corresponding GAPDH and related total protein expressions. The values are the percentage of the corresponding relative intensity of the control. Data are shown as the mean ± S.D. (n = 10). * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. 0 µg/ml SIPS-treated cells
Fig 5: RNA-seq data of HUVECs after stimulation with conidia of A. fumigatus. HUVECs were seeded in a six-well plate (2 × 106 cells per well) overnight before the conidia of A. fumigatus were added to the wells at a cell:spore ratio of 1:2. The cell lysates were harvested 2 and 6 h post-stimulation and sent for RNA sequencing. The heat-map (A) and volcano plots of 2 h (B) and 6 h (C) are shown. (D) The RNA expression of colony-stimulating factor 3 (CSF3) was analyzed by quantitative polymerase chain reaction (qPCR). *p < 0.05.
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