The Transcription Factor ZNF395 Is Required for the Maximal Hypoxic Induction of Proinflammatory Cytokines in U87-MG Cells

Figure 2

U87-MG cells were transfected with control siRNA or siRNA against ZNF395 and total RNA was isolated 48 hours after transfection (normoxia) or the cells were transferred 36 hours after transfection to 2% O₂ for additional 12 hours (hypoxia) before total RNA was isolated. cDNA was prepared and quantitative real time-PCRs were performed with primers specific for IL-6, IL-8, IL-1β, and LIF (a) and for MCP-1/CCL2, CA IX, and ZNF395 (b) RT-PCR of HPRT serving as internal control. The values obtained by dividing the Ct values for the cytokine and HPRT, respectively, from the siControl cells grown under normoxia were set as 1, and the fold activations were calculated. The standard deviations are given. Each PCR was performed six times in duplicate except that for ZNF395. (c) U87-MG cells were transfected with siControl or siZNF395; 24 hours later, one set of cells was incubated in the presence of DMOG, while the other set of cells obtained the equivalent amount of ETOH (which served as solvent for DMOG) as control. Another 24 hours later, the supernatant was used for ELISA to measure the level of IL-6 and IL-8. Each ELISA was performed two times in triplicate. QRT-PCR with RNA isolated from these cells was used to investigate the effect of DMOG on the expression of ZNF395. ; by Student’s -test.

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