Skip to main content
. 2015 Jan 29;6(1):e1621. doi: 10.1038/cddis.2014.591

Figure 2.

Figure 2

MKK3 depletion stabilizes wtp53 protein. Engineered MCF7 (a) and HCT116 (b) -sh/scr and -sh/MKK3 sublines were cultured with DOX (1.0 μg/ml) and collected at the indicated time points. Protein lysates (30 μg/lane) were analyzed by western blot analysis with anti-MKK3-, anti-p53-, anti-p21-, and anti-β-actin (loading control)-specific antibodies. Densitometry was performed with ImageJ software and relative p53 band intensity normalized to β-actin and quantified with respect to controls (sh-scr) set to 1.0. (c) Semi-quantitative RT-PCR was performed on total RNAs isolated from engineered MCF7 (left panel) and HCT116 (right panel) -sh/scr and sh/MKK3 sublines maintained 72 h with DOX. PCR was performed with specific set of primers. GAPDH was used as housekeeping gene. RT-PCR images were acquired by Bio-Rad Universal Hood II gel-imager. Densitometry was performed with ImageJ software and relative p53, p21, and MKK3 band intensity normalized to GAPDH and quantified with respect to controls (sh-scr) set to 1.0. (d) Upper panel: HCT116-sh/MKK3 sublines were transfected with sh/p53 or control sh/RNA carrying vector and efficiency of p53 depletion detected by western blotting (30 μg/lane). Lower panel: HCT116-sh/scr, -sh/MKK3-sh/RNA, and -sh/MKK3-sh/p53 were cultured 72 h with DOX, then total RNAs were isolated and semi-quantitative RT-PCR performed with set of primers specific to p21 and GAPDH (housekeeping gene). Images were acquired by Bio-Rad Universal Hood II gel-imager, and densitometry performed with ImageJ software. Relative p21 band intensity was normalized to GAPDH and quantified with respect to controls (sh-scr) set to 1.0. (e) Engineered H1299-sh/scr and -sh/MKK3 sublines were cultured with DOX (1.0 μg/ml) and collected 96 h later. Protein lysates (30 μg/lane) were analyzed by western blot analysis with anti-MKK3-, anti-p21- and anti-β-actin (loading control)-specific antibodies. Densitometry was performed with ImageJ software and relative p21 and MKK3 band intensity normalized to β-actin and quantified with respect to controls (sh-scr) set to 1.0