Figure 6.
ATF4 is involved in UPR-mediated angiogenic factor production. Stable cell lines 81B-scshRNA, 81B-shATF4-5 and 81B-shATF4-9 were established with lentiviral vectors and treated without (NT) or with GD (2 mM) for 24 hours. A, Grp78 and ATF4 were assessed with western blot and q-PCR. B, angiogenic factor expression was quantified with q-PCR. C, secreted VEGF was quantified with ELISA. D, UM-SCC-81B cells were treated with GD (2 mM) for 18 hours and processed for ChIP assay. PCR products of VEGF promoter fragments (AsnSyn site), asparagine synthetase (NSRE-1 site) and GAPDH genes were resolved in 1% agarose gel and stained with SYBR Green. The input was obtained by amplification of the initial unfractionated cell extracts. VEGF signal was quantified and normalized to GAPDH. *: p < 0.05.