Nontransformed rat intestinal epithelial IEC-6 cells (ATCC CRL-1592) were cultured as described before (14 (link)). Experimental procedures were described in Text S1 and Fig. S1A . At the end of the treatments, in-cell Western (ICW) procedure and immunofluorescence analysis were performed to analyze DNA damage via phosphorylation of the histone H2AX.
The ICW procedure was performed as previously described (53 (link)). The cells were fixed, permeabilized, blocked, and then incubated overnight (ON) with rabbit monoclonal anti-γH2AX 1/200 (20E3; Cell Signaling, Saint-Quentin en Yvelines, France). An infrared fluorescent secondary antibody (IRDye 800CW; Rockland) (1/500) was applied simultaneously with RedDot2 (1/500) (Biotium, Interchim, Montluçon, France) for DNA labeling. The DNA and γH2AX were visualized using an Odyssey infrared imaging scanner (LI-COR Science Tec, Les Ulis, France). All experiments were carried out in triplicate.
The ICW procedure was performed as previously described (53 (link)). The cells were fixed, permeabilized, blocked, and then incubated overnight (ON) with rabbit monoclonal anti-γH2AX 1/200 (20E3; Cell Signaling, Saint-Quentin en Yvelines, France). An infrared fluorescent secondary antibody (IRDye 800CW; Rockland) (1/500) was applied simultaneously with RedDot2 (1/500) (Biotium, Interchim, Montluçon, France) for DNA labeling. The DNA and γH2AX were visualized using an Odyssey infrared imaging scanner (LI-COR Science Tec, Les Ulis, France). All experiments were carried out in triplicate.
Free full text: Click here
