Figure 7. Knockdown of A20 induces the apoptosis of MDSCs through JNK pathway.
(a) Isolation of MDSCs from tumor tissue was confirmed by flow cytometry. Total number of 30000 cells was analyzed. Numbers illustrated indicate the percentage of the cells in total cells (n = 5). (b) mRNA levels of A20 from the isolated MDSCs from spleen and E.G7 tumor were measured (n = 3). (c) Fluorescence immunostaining of Gr1 and A20 positive cells in MDSCs isolated from tumor tissue. Scale bar 50 μm. (d) Si-A20 treatment induced the apoptosis of MDSC in vitro. Gr1+CD11b+ cells were isolated from tumors in E.G7 tumor-bearing mice and transfected with si-RNA. Apoptosis of MDSCs were confirmed by flow cytometry. Total number of 30000 cells was analyzed. Numbers illustrated indicate the percentage of the cells in total cells. MDSCs were cultured with a mixed medium (TCCM: complete RPMI 1640 = 1:1) with GM-CSF (10 ng/ml). Percentages of the cells in the regions were illustrated. (e) Statistic analysis of (d). (f) Si-A20 induced the apoptosis of MDSCs through JNK pathway. p-JNK, activated caspase-3 and activated caspase-8 were analyzed by western blotting after treatment of MDSCs with si-A20 and TNF-α. Data were representative of two independent experiments. Data represent means ± SD. *p < 0.05 (ANOVA test, Student’s t-test).