Effects of bacterial genotoxin on P62/SQSTM1 and UNR/CSDE1 localization.
A) Images of SK-Hep-1 cells following a 72 h coculture with colibactin-producing Escherichia coli. SK-Hep-1 cells were non-infected or infected for 3 days with colibactin-secreting extra-intestinal pathogenic E. coli and its corresponding isogenic mutant. Then, cells were processed for fluorescent staining with primary antibodies generated against P62/SQSTM1 (red) and UNR/CSDE1 (green) associated with fluorescent labeled-secondary antibodies and DAPI to counterstain the nuclei (blue). SK-Hep-1 cells non infected and infected with E. coli that don’t secretes colibactin did not show increase P62/SQSTM1 bodies. Thus, only SK-Hep-1 cells infected with colibactin-secreting E. coli are shown. (A1) SK-Hep-1 cells with distended nucleus without NR formation. (A2) and (A3) SK-Hep-1 cells with distended nucleus with NR formation. Arrows indicate nucleoplasmic reticulum (NR). Scale bar, 30 μm. As previously demonstrated, NR formation is primarily observed in response to bacterial genotoxin, CDT and colibactin [6]. Thus, images of non-infected cells and cells infected with colibactine-defective mutant strain are not presented. (B) In vivo detection of nuclear remodeling following a 14 months infection with H. hepaticus. Non-transgenic mice were infected with H. hepaticus wild type strain 3B1for 14 months [23]. Mice livers were processed and stained as previously reported [6,23]. Image of tissue sections of infected liver stained with primary and fluorescent secondary antibodies: UNR (red), P62/SQSTM1 (green) and DAPI to counterstain the nuclei (blue). The zone without UNR/P62/DAPI staining in the core of NR probably corresponds to a mitochondrion, as previously reported [6]. P62/SQSTM1-labeling generated a significant background noise in the mice liver tumor infiltrates, preventing efficient quantification. As previously demonstrated, NR formation is primarily observed in response to CDT intoxication, via its active CdtB subunit [6]. Thus, images of non-infected mice are not presented below. Arrows indicate nucleoplasmic reticulum (NR) enclosing UNR and P62/SQSTM1. Scale bar, 20 μm. Abbreviations: DAPI, 4′, 6′-diamidino-2-phenylindol; P62, P62/SQSTM1.
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