Hypoxia Potentiates Glioma Immunosuppression Figure 4. Hypoxia increased the intracellular pSTAT3 and HIF-1a expression in gCSCs. A and B. The pSTAT3 expression in the gCSCs was measured by intracellular pSTAT3 (pY705) staining via flow cytometry. The mean fluorescent intensity of positive staining was determined by flow cytometry analysis under both normoxic and hypoxic conditions for each gCSC (representative histogram was shown as A), and then the percentage change was calculated compared with the expression of pSTAT3 in each gCSC under normoxic condition (shown as B). C and D. The HIF-1a expression in the gCSCs was measured by intracellular HIF-1a staining via flow cytometry after culture in either hypoxic or normoxic culture conditions: representative histogram was shown as B, and percentage changes of all 4 gCSCs under hypoxia compared to normoxic condition was plotted as bar graphs in D. E. Confirmatory representative western blot demonstrating that pSTAT3 and HIF-1a expression is increased within gCSCs after hypoxia exposure. There was no change for total STAT3 expression. doi:10.1371/journal.pone.0016195.g004 PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16195