Figure 1

Torin-1 administration and genetic deletion of mTOR contributes to elevated differentiation of eosinophils. (a) Bone-marrow derived eosinophils (BMDE) during culture processes were harvested and mTOR (displayed as p-S6) was detected by western blot assay. (b) BMDE culture in vitro. Non-adherent mononuclear cells from bone marrow were obtained and differentiation was conducted with rmFlt-3L, rmSCF, and rmIL-5. Western Blot are performed according to auto-exposure and the resolution of 169 μm. Given that the highest level of p-S6 expression was on day 8, torin-1 or rapamycin was treated on day 8. (c) Blots were cropped from the same gel.) Mature eosinophils underwent Wrights-Giemsa staining after being cytospined. Images were acquired from an Olympus BX51 microscope (x4/0.3 NA objective with Olympus DP70 digital camera) for acquisition of images. (d,e) Percentage of eosinophils after rapamycin treatment (50 nM) was detected by flow cytometry (d) and statistically analysed as means ± SEM using Student’s T-test (e). (f,g) Percentage (g) of eosinophils after torin-1 administration (250 nM) was detected by flow cytometry and statistically analysed as means ± SEM (g). (h,i) Adenovirus with Cre recombinase was harvested from HEK293T cells and the titer measurement was undertaken. Bone-marrow-derived cells were infected with adenoviral administration 6 hours without serum. Percentage (h) of eosinophils was detected by flow cytometry and statistically analysed as means ± SEM (i). All the results above were triplicated by three independent experiments. Efficiency of adenoviral knockdown (j) Blots were according to different gels.) was detected on day 9. *P < 0.05, **P < 0.01, ***P < 0.001.