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

(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).

(A) 将MiaPaCa-2细胞转染靶向Nupr1的小干扰RNA（siNupr1）或靶向MSL1的小干扰RNA（siMSL1），随后在常规培养基中继续培养24小时；采用实时荧光定量反转录PCR（qRT-PCR）检测Nupr1与MSL1的mRNA表达水平，采用蛋白质印迹分析（western blot）检测蛋白表达水平，以β微管蛋白（β-tubulin）作为上样内参。(B) 将MiaPaCa-2细胞接种于盖玻片上，单独或共转染siNupr1与siMSL1，24小时后用10 µM顺铂（cisplatin）处理24小时。通过免疫荧光法进行γ-H2AX染色，采用40倍物镜计数γ-H2AX焦点数量。统计结果为10个视野的平均值，每个视野计数不少于100个细胞核（* p≤0.05）。(C) Nupr1与MSL1的邻近连接试验（Proximity Ligation Assay, PLA）：将细胞接种于盖玻片上，转染pcDNA3-Nupr1-Flag与pcDNA4-MSL1-V5重组质粒。实验次日，用5 µM或10 µM顺铂处理细胞以诱导DNA损伤，24小时后按照材料与方法部分所述步骤开展邻近连接试验。红色焦点代表Nupr1与MSL1的相互作用，仅转染pcDNA3-Nupr1-Flag质粒的细胞作为阴性对照。(D) Nupr1与MSL1不在DNA损伤位点发生相互作用：按照(B)中的方法重复邻近连接试验，随后进行γ-H2AX染色。采用40倍物镜的20个视野计数γ-H2AX绿色焦点、邻近连接试验红色焦点以及共定位的红绿焦点数量。统计结果为20个视野的平均值，每个视野计数不少于100个细胞核（* p≤0.05）。(E) 将MiaPaCa-2细胞接种于六孔板中，用30 µM顺铂处理24小时，采用细胞计数法按照材料与方法部分所述步骤评估细胞存活率，结果以对照组百分比表示。另将细胞接种于九十六孔板中，按照前述方法用顺铂处理后，采用Apo-ONE®均相半胱天冬氨酸蛋白酶3/7检测试剂盒检测半胱天冬氨酸蛋白酶3/7（caspase 3/7）活性；以CellTiter-Blue®细胞活力检测试剂盒按照厂商说明书进行归一化校正（* p≤0.05）。