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. 2015 Apr 30;5:9802. doi: 10.1038/srep09802

Figure 4. SMIFH2 downregulates mDia2, p53 and p300 protein levels.

Figure 4

(A) SMIFH2 induces the downregulation of mDia2, p53 and p300 in 293T cells. 293T cells were treated with DMSO or SMIFH2 for five hours. Total cell lysates (30 μg) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. One of two experiments that were performed with similar results is shown. (B-E) Proteasome inhibition fails to restore p53 levels during SMIFH2 treatment. (B) 293T, (C) A375, (D) U2OS, or (E) MDA-MB-231 cells were treated with DMSO (DMSO), SMIFH2 (SMIFH2), Lactacystin (Lact.), or SMIFH2 in combination with Lactacystin (SMIFH2 + Lact.) for five hours except for the A375 cells, which were treated for 2.5 hours. Total cell lysates (30 µg) were immunoblotted as indicated. One of two experiments that were performed with similar results is shown. (F) Quantification of mDia2 and p53 downregulation by SMIFH2. The expression of mDia2 and p53 were quantified by densitometric analyses of non-saturated films. For each of the indicated cell lines, intensities obtained in the SMIFH2- and DMSO-treated sample were normalized with respect to actin. Ratio between the normalized SMIFH2- and DMSO-treated intensities is represented as mean and SEM of at least three independent experiments. (G) Two different anti-p53 antibodies show that SMIFH2 reduces p53 levels. U2OS cells were treated with either DMSO or SMIFH2 for 4 and 5 hours (h = hours). Total cell lysates (30 μg) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Mouse monoclonal and rabbit polyclonal anti-p53 antibodies recognizing the region encompassing amino-acids 11-25 and 50-100 of p53, respectively, were used. One of two experiments that were performed with similar results is shown. A-E and G: Gels were run under the same experimental conditions and blots were cropped for final display.