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Figure 5 | Molecular Medicine

Figure 5

From: The p53 Upregulated Modulator of Apoptosis (PUMA) Chemosensitizes Intrinsically Resistant Ovarian Cancer Cells to Cisplatin by Lowering the Threshold Set by Bcl-xL and Mcl-1

Figure 5

PUMA chemosensitizes ovarian cancer cells through downregulation of Bcl-xL and Mcl-1. (A) A2780s and SKOV3 cells were subjected to the indicated treatments as described in Materials and Methods. Expression changes of Bcl-xL, Bcl-2 and Mcl-1 were analyzed by Western blotting. GAPDH was used as a loading control. Individual protein levels were measured by densitometric analysis of the Western blots and compared with GAPDH levels. Relative amount of individual proteins from control group cells was set as 1. (B) A2780s and SKOV3 cells were treated with cisplatin (5 µg/mL) for 24 h and analyzed for the expression of Bcl-xL and Mcl-1 by Western blotting. GAPDH was used as a loading control. Bcl-xL and Mcl-1 levels were measured by densitometric analysis of the Western blots and compared with GAPDH levels. A relative amount of Bcl-xL and Mcl-1 from untreated A2780s cells was considered as 1. (C) Semiquantitative RT-PCR was done for Bcl-xL, Mcl-1 and the control GAPDH (25 cycles) using the cDNA prepared from untreated or pcDNA3.1/hPUMA-treated A2780s and SKOV3 cells. (D) Real-time RT-PCR was carried out with Bcl-xL and β-actin-specific primers to quantify Bcl-xL mRNA levels. Columns, ratio of Bcl-xL mRNA relative to β-actin in untreated or pcDNA3.1/hPUMA-treated A2780s and SKOV3 cells. Bars, SD of each sample measured in triplicate (*P ≤ 0.05). (E) Both A2780s and SKOV3 cells were untreated or treated with pcDNA3.1/hPUMA in the presence or absence of zVAD-fmk (10 µmol/L) and MG132 (20 µmol/L). GAPDH was used as a loading control. hPUMA and Mcl-1 levels were measured by densitometric analysis of the Western blots and compared with GAPDH levels. Relative amount of hPUMA and Mcl-1 from untreated cells was considered as 1. zVAD-fmk almost completely blocked the disappearance of Mcl-1. The proteasome inhibitor MG132 also partially protected Mcl-1 from decay.

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