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. 2005 Sep 8;24(19):3470–3481. doi: 10.1038/sj.emboj.7600777

Figure 7.

Figure 7

Expression of a dn-PERK allele in human tumor cells decreases hypoxia tolerance and inhibits tumor growth. (A) Immunoblot of myc-tagged PERKΔC, eIF2α phosphorylation and β-actin (loading control). Exposure of HT29.Puro cells to extreme hypoxia induces an increase in eIF2α phosphorylation, which is blocked in HT29.PERKΔC cells. (B) Immunoblot of uncleaved and cleaved PARP in HT29.Puro and HT29.PERKΔC cells following hypoxia or 12 h treatment with 500 nM thapsigargin (top). β-Actin was used as a loading control. Apoptotic morphology following extreme hypoxia for 48 h at × 40 magnification (bottom). Arrowheads indicate cells with membrane blebbing and arrows indicate cells exhibiting vacuoles, a sign of ER stress. (C) PERK status does not affect induction of HIF-1α levels. HT29.Puro and HT29.PERKΔC cells were treated with extreme hypoxia or DFO. HIF-1α levels were determined by immunoblotting using nuclear extracts. (D) Tumor xenografts from HT29.Puro cells (left mouse) grow larger compared to tumors from HT29.PERKΔC cells (right mouse). (E) Growth measurements of HT29.Puro xenografts (blue line and squares) and HT29.PERKΔC xenografts (green line and triangles). Growth from six tumors was monitored over a period of 25 days following injection (mean±s.e.) (*P<0.05). (F) Immunoblot analysis of PERKΔC expression, eIF2α phosphorylation status or total eIF2α levels from excised and homogenized tumors shown in (D, E). (G) Sections from HT29.Puro and HT29.PERKΔC xenografts were stained for regions of hypoxia using a Cy-3-labeled anti-EF-5 antibody and apoptosis using an anti-cleaved caspase-3, FITC-labeled antibody. The merged images are shown on the right.