Figure 6

Nicotinamide suppresses DNA repair response. (a) Immunofluorescence staining of MDA-MB-231 cells incubated with 0 or 25 mM nicotinamide for 48 h. Green indicates γ-H2AX (H2AFX) foci, and blue indicates nuclei stained with Hoechst 33342. (b) Expression of γ-H2AX (H2AFX) in nicotinamide-treated cells were examined via western blotting. (c) Densitometry analysis of γ-H2AX (H2AFX) was quantitated using ImageJ. Values were normalized to the levels of β-actin. The data represent the mean ± SD (*P < 0.05). (d) Cytoscape visualization of cell cycle regulatory network. The colors of the border and inside circle indicate the expression levels of transcriptome and proteome data, respectively. Gray edge indicates the relationship of interacted/binding proteins based on the STRING database, whereas a strong solid and dotted line represent the relationships between proteins that are directly and indirectly affected, respectively. The arrows and inhibition symbols represent the activation and repression information. Node with asterisk mark indicates that the protein was assessed by only western blotting without the use of proteome data. There is no significant difference regarding the expression level if the color of the node is gray. Blue circle clustering highlights the complex enzymes of proteins. (e) Proposed model of nicotinamide-induced DNA repair interaction network. Networks are represented to reflect proteome, transcriptome and western blotting. Red indicates increased expression, blue indicates decreased expression and gray indicates no significant difference. (f) Expression of ATM, PARP1 and ATR in nicotinamide-treated cells was examined via western blotting. (g) Densitometry analysis of ATM, PARP1 and ATR was quantitated using ImageJ. Values were normalized to the levels of β-actin. Densitometric analysis was performed using ImageJ. The data represent the mean ± SD (*P < 0.05, **P < 0.01). Abbreviations: p - phosphorylation, WB - western blot.