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. 2013 Feb 26;15(5):562–577. doi: 10.1093/neuonc/not005

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© The Author(s) 2013. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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Fig. 6. — Chemosensitization to TMZ by P4HB inhibition activates ER stress-induced apoptosis via UPR signaling. (A) P4HB inhibition induced apoptosis. Flow cytometry analysis performed by double staining of annexin V and propidium iodide shows increased apoptotic response in D54-R and U87-R cells after P4HB inhibition by siRNA and/or TMZ treatment at 500 and 1000 µM. A representative experiment of 3 performed was shown. (B) Caspase-3/7 activity was measured by the Caspase-Glo 3/7 assay kit. Compared with the untreated and negative control groups, an increase in caspase-3/7 activity was observed in the shP4HB group and shP4HB + TMZ group. *P = .05; **P = .01. Data are means of triplicate experiments. Western blot depicting alterations in (C) the expression of apoptosis-related proteins in whole-cell lysates and (D) mitochondrial and cytosolic fraction of the release of cytochrome c (Cyto-c). COX IV was used as loading control for mitochondrial fraction and β-actin for cytosolic fraction. (E) Activation of UPR signaling was illustrated by representative blots of altered ER chaperones (GRP78) and stressor (PERK) expression prepared from cells of different groups (Scr-Ctrl + TMZ; D54-R: 0, 300 and 600 µM; U87-R: 0, 500 and 1000 µM) and shP4HB + TMZ (D54-R: 0, 300 and 600 µM; U87-R: 0, 500 and 1000 µM) for 72 h. COX IV was used as loading control. (F) Induction of ER stress markers (ATF4 and GADD34) was quantified by qPCR markedly in both D54-R shP4HB and U87-R shP4HB after TMZ treatment at our previously defined optimal concentration (300 and 600 µM; 500 and 1000 µM, respectively). Column, mean of 3 replicates ± SD. *P = .05; **P = .01.

Fig. 6. — Chemosensitization to TMZ by P4HB inhibition activates ER stress-induced apoptosis via UPR signaling. (A) P4HB inhibition induced apoptosis. Flow cytometry analysis performed by double staining of annexin V and propidium iodide shows increased apoptotic response in D54-R and U87-R cells after P4HB inhibition by siRNA and/or TMZ treatment at 500 and 1000 µM. A representative experiment of 3 performed was shown. (B) Caspase-3/7 activity was measured by the Caspase-Glo 3/7 assay kit. Compared with the untreated and negative control groups, an increase in caspase-3/7 activity was observed in the shP4HB group and shP4HB + TMZ group. *P = .05; **P = .01. Data are means of triplicate experiments. Western blot depicting alterations in (C) the expression of apoptosis-related proteins in whole-cell lysates and (D) mitochondrial and cytosolic fraction of the release of cytochrome c (Cyto-c). COX IV was used as loading control for mitochondrial fraction and β-actin for cytosolic fraction. (E) Activation of UPR signaling was illustrated by representative blots of altered ER chaperones (GRP78) and stressor (PERK) expression prepared from cells of different groups (Scr-Ctrl + TMZ; D54-R: 0, 300 and 600 µM; U87-R: 0, 500 and 1000 µM) and shP4HB + TMZ (D54-R: 0, 300 and 600 µM; U87-R: 0, 500 and 1000 µM) for 72 h. COX IV was used as loading control. (F) Induction of ER stress markers (ATF4 and GADD34) was quantified by qPCR markedly in both D54-R shP4HB and U87-R shP4HB after TMZ treatment at our previously defined optimal concentration (300 and 600 µM; 500 and 1000 µM, respectively). Column, mean of 3 replicates ± SD. *P = .05; **P = .01.