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. 2015 May 11;35(2):228–240. doi: 10.1038/onc.2015.76

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Copyright © 2016 Macmillan Publishers Limited

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MDM4 regulates HIPK2 protein levels. (a) Western blot (WB) of the indicated proteins in MCF10A cells transfected with 20 nm stealth MDM4-specific (siMDM4) or stealth control RNA (siCTL) for 48 h and exposed to 2 μm Adr for the indicated time points. Ratio of densitometric values of p53Ser46P to p53 total levels is shown at the bottom of the blot. p53Ser46P/p53Tot ratio in siCTL-treated cells was arbitrarily set to 1. (b) Upper panel: WB of the indicated proteins in p53^+/+ and p53^−/−HCT116 cells transfected with siCTL or siMDM4. Ratio of densitometric values of HIPK2 to tubulin is shown at the bottom of the blot. HIPK2/TUB ratio from control lane was arbitrarily set to 1. Lower panel: RTqPCR of HIPK2 mRNA normalized to hTBP. HIPK2 mRNA expression levels of siCTL cells were arbitrarily set to 1. (c) WB of the indicated proteins in MCF10A-tet-shMDM4 cells treated or untreated for 48 h with doxycycline, and collected at 24 h after infection with Adenoviral vector carrying MDM4 cDNA (AdMDM4) or an empty Adenoviral vector. Analysis of HIPK2 levels as in b. (d) WB of the indicated proteins in Mdm4^−/−p53^−/− MEFs transfected with the indicated expression vectors. Analysis of HIPK2 levels as in b. (e) WB of the indicated proteins in MCF10A cells transfected with siCTL or siMDM4 and treated with 25 μm Mg132 for 8 h. Analysis of HIPK2 levels as in b. (f) HCT116 cells transfected for 48 h with GFP-HIPK2 alone (CTL) or in combination with pcDNA3.1-MDM4 and treated with cycloheximide (CHX, 50 μg/ml). WCE were collected at the indicated time points and analysed by WB (left panel). Densitometric analysis of GFP-HIPK2 levels is shown in the right panel. HIPK2 bands intensity was normalized to Tubulin, then normalized to the t=0 value, set to 100%. (g) U2OS cells transfected as in a and at 24 h treated with cycloheximide (CHX, 70 μg/ml). WCE were collected at the indicated time points and analysed by WB (left panel). Analysis of HIPK2 levels (right panel) as in f.