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. 2009 Mar 10;100(7):1154–1164. doi: 10.1038/sj.bjc.6604969

Figure 2.

Figure 2

Induction of autophagy in U87MG cells treated with TMZ followed by CRAd-S-pk7 infection. Effect of combined treatment of U87MG cells was detected by (A) light microscopy, (B) western blot, (C) membrane potential, (D) flow cytometry and (E) LDH toxicity. (A) Decrease in cell density and morphological changes associated with treatment with TMZ followed by CRAd-S-pk7. (B) Modulation of pro-apoptotic, anti-apoptotic and autophagic proteins in response to treatment with TMZ (100 μM), CRAd-S-pk7 (100 vp per cell) or combination (TMZ and CRAd-S-pk7) as determined by Western blot analysis. Pretreatment of U87MG cells with TMZ followed by CRAd-S-pk7 infection showed over-expression of p53, Bax and APG5 proteins and downregulation of Puma, Noxa, BNIP3 and BCL-2 proteins. There was no evidence of cleaved Caspase-3. (C) To show that mitochondrial pathway is not activated, we measured mitochondrial potential changes. TMZ, CRAd-S-pk7 and combination group did not induce significant changes in Δψ. (D) Autophagy was determined by staining with acridine orange (AO) and α-LC3B antibody followed by flow cytometry analysis. Experiment was performed in triplicates and the mean of two independent experiments is shown here. (E) Effect of Bafilomycin A1 (BAF-A1) and 3-MA treatments on co-treatment induced toxicity. Cells were pretreated with 3-MA, BAF-A1 or vehicle control for 12 h before exposure to TMZ, CRAd-S-pk7 or TMZ followed by CRAd-S-pk7. Figure summarises data from two independent experiments each having six replicates per condition. (*), (**) and (***) P<0.05. (*), (**) and (***) P-value determined by comparing mock vs TMZ alone, CRAd-S-pk7 alone or combination TMZ then CRAd-S-pk7, respectively.