Figure 2
Protein detection for CD133−/CD15−/NESTIN− glioma cells under different oxygen levels. (A) Higher MGMT and ABCG2 levels were detected for GL261 CD133−CD15−NESTIN− cells under 1%O2 conditions for 24 h than 21%O2 or 95%O2 via immunofluorescence staining. Cells demonstrated the lowest expression in 95%O2 (P < 0.05, One-way ANOVA). (B) A 5-fold increase of MGMT in 1%O2 was identified for GL261 CD133−CD15−NESTIN− cells via RT-qPCR compared with normoxia, and MGMT was fewer in 95%O2. Similar results were obtained for ABCG2 in different oxygen levels (P < 0.05, One-way ANOVA). (C) There were significant differences in MGMT and ABCG2 for GL261 CD133−CD15−NESTIN− cells under different oxygen levels in western blot; the highest expression was identified in 1%O2, and the lowest expression was demonstrated in 95%O2 (P < 0.05, One-way ANOVA). Both MGMT and ABCG2 increased levels in a time dependent manner from hypoxia for 24 to 48 h. However, under hyperoxia, MGMT expression remained stable; for ABCG2, there was lower expression after hyperoxia treatment (P < 0.05, One-way ANOVA). (D) Compared with 21%O2 or 95%O2, increased CD133, CD15 and NESTIN levels were identified for GL261 CD133−CD15−NESTIN− cells in 1%O2 via immunofluorescence staining. Cells demonstrated the lowest expression after exposure in 95%O2. In contrast, GFAP was highly expressed after hyperoxia exposure for 24 h, which exhibited 3-fold increases compared with normoxia and 12-fold compared with hypoxia. There was almost no GFAP expression in the 1%O2 or 21%O2 microenvironments (P < 0.05, One-way ANOVA). (E) RT-qPCR indicated the CD133, CD15 and NESTIN levels significantly increased in 1%O2 for 12 h compared with 21%O2 and 95%O2 for GL261 CD133−CD15−NESTIN− cells. However, the GFAP levels increased in an oxygen concentration-dependent manner, and GFAP was 3-fold higher under hyperoxia than normoxia and 6-fold higher than hypoxia. A significant difference was also identified between the 1%O2 and 21%O2 microenvironments (P < 0.05, One-way ANOVA). (F) GL261 CD133−CD15−NESTIN− cells presented the highest expression of CD133, CD15 and NESTIN after 1%O2 exposure for 24 or 48 h. Furthermore, CD133, CD15 and NESTIN increased levels in a time-dependent manner from hypoxia for 24 to 48 h; however, under 21%O2 conditions, the expression levels significantly decreased for CD133 and NESTIN in a time-dependent manner. Under 95%O2, CD133, CD15 and NESTIN were the lowest expression. In contrast, GFAP was highly expressed in 21%O2 and 95%O2 with lower detection under 1%O2 conditions. Furthermore, the GFAP levels increased in a time-dependent manner from normoxia or hyperoxia for 24 to 48 h (P < 0.05, One-way ANOVA).
