Figure 1.

Decrease of Stau2 expression in response to camptothecin (CPT) treatment. (A–C) HCT116 cells were treated for 24 h with increasing doses of CPT as indicated and cell extracts were analyzed by western blotting (A) and RT-qPCR (C). (B) Quantification of Stau2⁵² protein and mRNA (all isoforms) levels and of PARP1 cleavage (PARP1⁸⁹/PARP1¹¹⁶) in the representative experiment. The western blot is representative of three independently preformed experiments. PARP1 cleavage was used as a measure of apoptosis and β-actin as a loading control. The RT-qPCR data represent the means and standard deviation of three independently performed experiments. The ratio of specific gene mRNAs on GAPDH mRNA in DMSO-treated cells (0 nM) was arbitrary fixed to 1. Stau1, Staufen 1 (an RNA-binding protein—paralog of Stau2); APAF1, apoptotic peptidase activating factor 1; GRP78, glucose-related protein 78. APAF1 mRNAs, known to be upregulated in apoptotic cells, was used as positive controls. GRP78 was used as a negative control. Statistical analyses (Student's t-test) are indicated when significant. ***P-value ≤ 0.001; **P-value ≤ 0.01; *P-value ≤ 0.05. (D–F) HCT116 cells were incubated in constant amounts of CPT (300 nM) for increasing periods of time. Cells were lysed and Stau2 expression was analyzed by western blotting (D) and RT-qPCR (F). (E) Quantification of Stau2 protein and mRNA levels and of PARP1 cleavage (PARP1⁸⁹/PARP1¹¹⁶) over times in the representative experiment. The thin black lines represented the best fit on the curves. The western blot is representative of four independently performed experiments. Stau2 decline and PARP1 cleavage were normalized to their values at time 0. The RT-qPCR data represent the means and standard deviation of four independently performed experiments. The ratio of specific gene mRNAs on GAPDH mRNA at time 0 was arbitrary fixed to 1.