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

Figure 1

RTS3b, C33A, HEK293, U87-MG, and U937 cells were either grown under ambient O₂ atmosphere or in the presence of 2% O₂ for 12 hours before total RNAs or total protein extracts were prepared. (a) Quantitative real time RT-PCR was performed with each RNA and primers specific for ZNF395 and HPRT, which served as house-keeping gene. The ZNF395 values for RTS3b grown at ambient atmosphere were arbitrary set as 1 and the fold activations obtained for all other cells were calculated according to the comparative threshold method as previously described [27]. (b) A Western blot with 60 μg protein extracts from each cell line grown under normoxia or hypoxia was developed with antibodies against HIF-1α, ZNF395, or actin, which served as loading control. (c) U87-MG cells were either left untransfected (lanes 1, 2) or transfected with control siRNA (lane 3) or siRNA against HIF-1α (lane 4). 36 hours later, the cells were set to hypoxia for 12 hours, as indicated, before protein extracts were prepared and a Western blot with antibodies against ZNF395 and actin was performed. One set of cells was transfected with siControl or siHIF-1α and 48 hours later total RNA was isolated, which was used to perform qRT-PCR with primers for HIF-1α and HPRT. The fold activation was calculated as in (a).

(a)

(b)

(c)