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Figure 4 | Journal of Experimental & Clinical Cancer Research

Figure 4

From: Sp1 is involved in H2O2-induced PUMA gene expression and apoptosis in colorectal cancer cells

Figure 4

Sp1 is required for the transactivation of the human PUMA promotor by H 2 O 2 . (A) LoVo cells were transiently transfected with reporter plasmids (0.5 μg). 0.64 mM H2O2 was added 24 hours post-transfection for 15 minutes. The pSVβ-Galactosidase plasmid (0.1 μg) was included in each sample for normalization of transfection variability. Luciferase activity was determined in cell lysates 48 hours following the transfection and the values (mean ± SD), from at least two independent experiments performed in duplicate (*p > 0.05,-336/+157 -126/-25PUMA-Luc vs-136/+157 PUMA-Luc, #p < 0.05,-36/+157PUMA-Luc vs-336/+157 -126/-25PUMA-Luc). (B) Mithr.A (200 ng/ml) was added to LoVo cells. Extracts from the treated or untreated LoVo cells were subjected to immunoblotting using antibodies against p53, Sp1 and actin. (C) LoVo cells were treated with 20 μM PFT-α and/or 200 ng/ml Mithr.A an hour before 0.64 mM H2O2 added. Cell extracts were analysed for the expression of endogenous p53, Sp1 and actin as indicated by immunoblotting. (D, E) LoVo and HCT 116 cells were treated with 20 μM PFT-α and/or 200 ng/ml Mithr.A an hour before 0.64 mM H2O2 added. Cell extracts were analysed for the expression of PUMA as indicated by immunoblotting. (F) H2O2 -induced PUMA expression was abrogated by transfecting Sp1 siRNA. LoVo cells were transfected with control siRNA or Sp1 siRNA and H2O2 was added 24 hours post-transfection. PUMA expression was detected by Western blotting analysis 48 hours post-transfection. (G) LoVo cells were transiently transfected with the -336/+157 PUMA-Luc reporter plasmid, 0.64 mM H2O2 was added 24 hours post-transfection and remained for 15 minutes. Mithr.A (200 ng/ml) and/or PFT-α (20 μM) was added an hour before H2O2. Luciferase activity was determined in cell lysates 48 hours following the transfection and the values (mean ± SD.) from at least two in dependent expression performed in duplicate are shown in the form of a bargraph, *p < 0.05, significant difference between cells treated as indicated in Figure 4G, # p > 0.05, no significant difference between cells treated as indicated in Figure 4G (factor analysis, n = 6).

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