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

Figure 4.

Figure 4

MKK3 depletion reduces mutp53 protein levels through autophagy. Engineered MDA-MB468 (a) and HT29 (b) -sh/scr and -sh/MKK3 sublines were cultured with DOX (1.0 μg/ml) and collected at indicated time points. Protein lysates (30 μg/lane) were analyzed by western blot analysis with anti-MKK3-, anti-p53-, 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) Markers of autophagy LC3-I, LC3-II, and p62 upon MKK3 depletion were assessed by western blotting in mutp53 MDA-MB468 (left panel) and HT29 (right panel) engineered -sh/scr and sh/MKK3 sublines. Seeded cells (1.5 × 105 cells/60 mm dish) were collected at 120 h post DOX delivery, and protein lysates (15 μg /lane) resolved in SDS–polyacrylamide gel electrophoresis (PAGE) and probed with anti-MKK3-, anti-LC3-, and anti-SQSTM1/p62-specific antibodies. Densitometry analyses were performed with ImageJ software and LC3-II band intensity normalized to β-actin and quantified with respect to control tumors (sh/scr) set to 1.0. (d) Engineered HT29-sh/scr and -sh/MKK3 sublines were seeded (1.5 × 105 cells/60 mm dish) and maintained in DOX condition for 72 h, then transfected with siRNA for ATG5 (si-ATG5) or with control siRNA (si-ctr). Forty-eight hours after transfection, cells were collected and protein lysates (15 μg /lane) resolved in SDS-PAGE and probed with anti-ATG5- and anti-SQSTM1/p62-specific antibodies. β-Actin was used as loading control. Densitometry analyses were performed with ImageJ software and anti-SQSTM1/p62 band intensity normalized to β-actin and quantified with respect to control tumors (sh/scr) set to 1.0. (e) MDA-MB468 sh/scr and sh/MKK3 sublines were cultured 120 h with DOX and, in the last 48 h, treated/untreated with CQ (25 μM). Afterwards, cells were collected and lysate analyzed by western blot analysis with anti-p53-, anti-MKK3-, and anti-β actin (loading control)-specific antibodies