|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 4. Both HRSL3-dependent PP2A inhibition and siRNA-mediated suppression of PP2A regulatory subunit A, induce apoptosis via a caspase-dependent pathway. (A) Cleaved caspase-3 was detected by immunoblotting using appropriate antibodies. Total amount of caspase-3 was used as a loading control. In all experiments HRSL3 expression was controlled by immunoblotting with the HRSL3 specific antibody. Application of the caspase inhibitor zVAD-fmk leads to the abrogation of caspase cleavage. (B) OVCAR-3 cells were electroporated with the HREV1FL, dNdC expression plasmids. Twelve hours after transfection, cells were treated with 40 µM caspase inhibitor zVAD-fmk, if indicated. Alternatively, cells were treated with OA (10 nM), OA with zVAD-fmk (40 µM), or vehicle DMSO only. Seventy-two hours after transfection or treatment, nuclear DNA content was measured by flow cytometry. The relative number of cells displaying an apoptotic, sub-G1 content, is given between the marker bars. DNA histograms are representative of two independent experiments. (C) OVCAR-3 cells were transfected with PR65
- and PR65
-specific siRNAs individually, together, and treated with caspase inhibitor zVAD-fmk (40 µM), if indicated. The level of the PR65 protein and cleavage of caspase-3 were controlled 48 hours after transfection (upper panel). Efficiency of the PR65-specific RNA interference was verified using RT-PCR 12 and 24 hours after transfection. As a control, GAPDH mRNA was amplified (lower panel). (D) OVCAR-3 cells were transfected with PR65- and PR65-specific siRNAs individually or in combination. Apoptosis was analyzed at the single-cell level by measuring the nuclear DNA content. The relative number of cells displaying a sub-G1 DNA content is given between the marker bars. DNA histograms are representative of three independent experiments.
|
