Effects of T310A point mutation on TSP50-mediated resistance to DOX-induced apoptosis.

<p>(A) pcDNA3 cells, pcDNA3-TSP50 cells, and pcDNA3-TSP50 T310A cells were treated with 5 µg/ml of DOX for 12 h, and cleaved-caspase 3 protein levels in these cells were detected by Western blotting. GAPDH was used as a loading control. (B) S-shRNA, TSP50 shRNA#1, and TSP50 shRNA#4 were transfected into pcDNA3-TSP50 cells, and the TSP50 mRNA levels of these cells were analyzed by RT-PCR. β-actin was used as an internal control to check the efficiency of cDNA synthesis and PCR amplification. (C) S-shRNA, TSP50 shRNA#1, and TSP50 shRNA#4 were transfected into pcDNA3-TSP50 cells, and cleaved-caspase 3 and TSP50 protein levels in these cells were detected by Western blotting after pretreatment with 5 µg/ml of DOX for 12 h. GAPDH was used as a loading control. Quantification of WB for TSP50 levels using ImageJ software is shown on the left panel. (D) Cells were treated with 5 µg/ml of DOX for 12 hours and stained using the TUNEL Assay Kit (Cat. no 12 156 792 910 Roche Applied Sciences, Mannheim, Germany).