qPCR analysis to determine fold change in mRNA expression of catalase in MDA-MB-468 cells at 4 h following treatment with 1 mM DETA-NONOate [a]. Immnoblot analysis of the expression of catalase in lysates of MDA-MB-468 cells following treatment with 1 mM DETA-NONOate [b, left panel].

<p>Densitometric analysis of catalase expression utilizing the Image Quant software [b, right panel]. Enzyme activity of catalase in 100 µg of cell lysate of MDA-MB-468 cells with or without the addition of 1 mM DETA-NONOate [c, left panel] Enzyme activity of catalase in 100 µg of cell lysate of MCF-7 cells with or without the addition of 1 mM DETA-NONOate [c, right panel]. Silencing of catalase in MDA-MB-468 cells utilizing siRNA against human catalase depicted by immunobloting and qPCR [d, left and right panel]. H₂O₂ production in MDA-MB-468 cells either transfected with scramble controls or siRNA against catalase, at 24 h following treatment with 1 mM DETA-NONOate [e]. Annexin V binding assay at 24 h, in MDA-MB-468 cells either transfected with scrambled controls [siScramble] or siRNA against the human catalase gene, following treatment with 1 mM DETA-NONOate [1 mM DETA] [f]. Graphical representation of the quantification of cells undergoing apoptosis in either scramble controls or in catalase silenced MDA-MB-468, following treatment with 1 mM DETA-NONOate [g]. Caspase-3 activity in MDA-MB-468 at 24 h following treatment with 1 mM DETA-NONOate in either scrambled controls [siScramble] or silenced for human catalase gene [siCAT] [h]. Results are expressed as the means from three independent experiments performed in duplicate; <i>bars</i>, SE wherever applicable. Western blot images are representative of three different experiments. GAPDH is probed as an equal loading control wherever applicable.</p

Immunoblot analysis of the dephosphorylation of pFOXO1 at serine 256, in MDA-MB-468 cell lysates, over time following treatment with 1 mM DETA-NONOate [a, left panel]. Immunoblot analysis of the dephosphorylation of pFOXO1 at serine 256 in MCF-7 cell lysates at 24 h post-treatment with 1 mM DETA-NONOate [a right panel].

<p>Immunoblot analysis of dephosphorylation of pFOXO1 upon DETA-NONOate treatment in either scrambled controls or MDA-MB-468 cells silenced for the catalytic subunit of PP2A [PP2A siRNA] [b]. Immunoblot analysis of FOXO1 in MDA-MB-468 nuclear fractions at 24 h post-treatment with 1 mM DETA-NONOate [c ]. Silencing of FOXO1 in MDA-MB-468 cells utilizing siRNA against human FOXO1 [d left panel] qPCR analysis to determine the fold change in mRNA expression of FOXO1 in MDA-MB-468 transfected with either scrambled controls [SiScramble] or siRNA against the human FOXO1 gene [siFOXO] [d, right panel]. Annexin V binding assay at 24 h, in MDA-MB-468 cells either transfected with scrambled controls [siScramble] or siRNA against the human FOXO1 gene, following treatment with 1 mM DETA-NONOate [1 mM DETA] [e]. Caspase-3 activity in MDA-MB-468 at 24 h following treatment with 1 mM DETA-NONOate in either scrambled controls [siScramble] or silenced for human FOXO1 gene [siFOXO] [f]. Results are expressed as the means from three independent experiments performed in duplicate; <i>bars</i>, SE wherever applicable. Western blot images are representative of three different experiments. GAPDH and Lamin A are probed as equal loading controls for cytosolic and nuclear nuclear fractions respectively.</p

qPCR analysis to compare the expression of catalase mRNA in MDA-MB-468 cells transfected with scrambled controls, siRNA against FOXO1 and SiRNA against PP2A following treatment with 1 mM DETA-NONOate for 4 h [a]. Immunoblot analysis of catalase protein expression in the lysates of MDA-MB-468 cells transfected with scrambled controls, siRNA against FOXO1 and siRNA against PP2A at 24 h following treatment with 1 mM DETA-NONOate [b].

<p>Immunoblot analysis of total and pFOXO1 in lysates of MDA–MB-468 cells at 24 h, following overexpression of either the human catalase gene [MDAOXCAT] or empty vectors in scrambled controls or in cells silenced for the catalytic subunit of PP2A [c–d]. Results are expressed as the means from three independent experiments performed in duplicate; <i>bars</i>, SE wherever applicable. Western blot images are representative of three different experiments. GAPDH is probed as an equal loading control wherever applicable.</p