Fig. 1: Hypoxic microenvironment improves tolerance of glioma cells to temozolomide though decreased DNA damage and protection mitochondria function.
a U87MG cells exposed to normoxic (20% O2) or hypoxic (1% O2) conditions were pretreated with TMZ of various concentration for 72 h and subjected to CCK8 Assay. b U87MG cells exposed to normoxic or hypoxic condition were pretreated with TMZ (250 μM) for indicated time points, then subjected to CCK8 Assay. c The apoptotic rates of U87MG cells exposed to normoxic or hypoxic condition upon treatment with DMSO or TMZ (250 μM) for 72 h were measured by flow cytometry. d Western blotting analysis showing levels of caspase-3, cleaved caspase-3, PARP, cleaved PARP, Bax, Bcl-2, and γH2AX after indicated treatment. e, f The cells with treatment as indicated were stained with JC-1 probe and detected by fluorescence microscope and flow cytometer. Red fluorescence represents the aggregate form of JC-1, indicating high mitochondrial membrane potential (ΔΨm). Green fluorescence represents the monomeric form of JC-1, indicating impaired mitochondrial membrane potential (ΔΨm). g Immunofluorescence staining of γH2AX in U87MG cells exposed to normoxia or hypoxia and TMZ (250 µM) or not for 72 h (magnification, ×400). Scale bar = 100 μm γH2AX: Green; DAPI: Blue. Data were presented by means ± SEM. in triple experiments. Asterisk indicated significant difference at P < 0.05 between normoxia group and hypoxia group. Double asterisk indicated significant difference at P < 0.01 between control group and TMZ treatment group in normoxia. Double hash indicated significant difference at P < 0.01 between normoxia group and hypoxia group treated with TMZ
