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. 2013 Oct 30;8(10):e78101. doi: 10.1371/journal.pone.0078101

Figure 1. Cell survival and caspase 3/7 activity in response to cisplatin treatment and Nupr1 and MSL1 interaction.

Figure 1

(A) MiaPaCa-2 cells were transfected with siNupr1 or siMSL1 and cultured in conventional media for an additional 24-h period; Nupr1 and MSL1 mRNA expression was measured by qRT-PCR, and proteins levels by western blot analysis. β-tubulin was used as a control of loading. (B) MiaPaCa-2 cells were plated on coverslips and transfected with siNupr1 or siMSL1, alone or together, and 24 h later treated with cisplatin (10 µM) for a 24 h-period. γ-H2AX staining was performed by immunofluorescence. The 40× magnification was used to count the number of γ-H2AX dots. Data are the means of 10 field counting with not less than 100 nucleus counted (* p≤0.05). (C) Proximity Ligation Assay (PLA) of Nupr1 and MSL1. Cells were plated on coverslips and transfected with pcDNA3-Nupr1-Flag and pcDNA4-MSL1-V5 constructs. The day after the experiment, cells were treated with cisplatin (5 µM or 10 µM) to induce DNA damage and 24 h later the PLA was performed as described in Material and methods section. Red dots represent Nupr1/MSL1 interaction. DNA transfection with only pcDNA3-Nupr1-Flag construct was used as a negative control. (D) Nupr1 and MSL1 do not interact into the DNA damage sites. PLA was reproduced as in (B) and followed by γ-H2AX staining. A 20 fields of 40× magnification were used to count the number of γ-H2AX green dots, the number of PLA red dots and the number of co-localizing green and red dots. Data are the means of 20 field counting with not less than 100 nucleus counted (* p≤0.05). (E) MiaPaCa-2 cells were plated in six-well plates and treated with cisplatin (30 µM) for 24 h. Cell survival rate was estimated using a cell counter as described in the Material and Methods section. Data are expressed as percentage of the control. Cells were plated in ninety-six-well plates treated with cisplatin as described above and caspase 3/7; we assayed the caspase acativity by using the Apo-ONE® homogenous caspase 3/7 assay. The normalization value was performed by using CellTiter-Blue® viability assay according to manufacturer’s instructions (* p≤0.05).