Fig 1

MDM4 inhibits S6K1 phosphorylation. a Representative Wb analysis of the indicated proteins in MCF10A cells transfected with siCTL or siMDM4 and collected after 48 h (hrs). Histogram in the lower panel shows the ratio of densitometric values of phosphorylated S6K1 (pS6K1) to S6K1. The ratio pS6K1/S6K1 from siCTL lane was arbitrarily set to 1. Mean ± SD of two independent biological replicates is shown (N = 2). b Wb analysis of the indicated proteins in p53^−/−HCT116 and p53^+/+HCT116 cells transfected as in (a). Histogram as in (a) (N = 2). c Wb analysis of the indicated proteins in 293 T cells transfected with siMDM4 or siCTL and after 24 h treated with Rapamycin (RAPA, 40nM) for additional 24 h. Histogram as in (a) (N = 2). d Wb analysis of the indicated proteins in 293 T cells transfected as in (c) and after 48 h treated with EBSS for 50’ and then in presence or absence of amino acids mixture (aa) for 30’, or with RAPA for 1 h. Histogram as in (a) (N = 2). e Wb analysis of the indicated proteins in 293 T cells transfected as in (c). After 48 h, cells were grown in medium deprived of amino acids (w/o aa) for 3 h, and in absence or presence of aa for the last 10’. Histogram as in (a) (N = 2). f Wb analysis of the indicated proteins in 293 T cells transfected with pcDNA3.1-MDM4 (MDM4) or control vector (CTL) and after 24 h treated as in (e) (the two panels derive from the same blot). Histogram as in (a). The ratio pS6K1/S6K1 from CTL lane was arbitrarily set to 1 (N = 2). g Wb analysis of the indicated proteins in Hela cells transfected with siMDM4 or siCTL and with shcontrol vector (Mock) or shmTOR for 48 h, then treated with EBSS for 50’, and for additional 30’ with aa. Histogram as in (a). (N = 3, * = p < 0.05, two‐tailed unpaired t‐test)