Fig. 2

ADAM17 levels during oncogene-induced senescence. a MCF7 Tet-Off p95HER2 cells were cultured in the presence or absence of doxycycline and harvested at the specified time points. Then cells were lysed and cell lysates were analyzed by Western blotting with the indicated antibodies. The densitometric quantification of the pro- and mature forms of ADAM17 and ADAM10 was performed in four independent experiments and is expressed as average ± standard deviation. *P <0.05, **P <0.01 using the Student’s t test. b Relative ADAM17 and ADAM10 mRNA expression was determined by reverse transcription-polymerase chain reaction in the same cells as in (a). Data were normalized to 18S mRNA. Averages and standard deviations of three independent experiments are represented. Differences are not significant statistically (ns). c MCF7 Tet-Off p95HER2 cells were cultured in the presence or absence of doxycycline for 7 days and incubated with phosphate-buffered saline (unlabeled) or sulfo-NHS-LC-biotin (biotinylated) as described in the Methods section. Then cells were lysed and cell lysates were incubated with anti-ADAM17 antibody and immunoprecipitates were analyzed by Western blotting by using streptavidin-peroxidase (Str.) or anti-ADAM17 (αA17). The densitometric quantification of six independent experiments is expressed as average ± standard deviation. **P <0.01 using the Student’s t test. d Representative immunofluorescence of ADAM17 (green) and DAPI (4’,6’-diamidino-2-phenylindole) counterstaining (nuclei, red) in MCF7 Tet-Off p95HER2 cells cultured with or without doxycycline. Bottom and left panels in each image represent Z-stack integration in the X and Y dimensions, respectively. e MCF10A Tet-On p95HER2 cells were cultured for 7 days without or with doxycycline. Then senescence-associated β-galactosidase was performed. Representative bright-field images are shown. f The same cells as in (e) were lysed and cell lysates were analyzed by Western blot with the indicated antibodies. Doxy doxycycline