Figure 2.

HEK 293 cells depleted of Penk by siRNA are protected from apoptotic cell death. (a–d) 293 cells were transfected with two siRNA constructs directed against Penk and a scrambled oligonucleotide as control (Penk 1, Penk 2 and Scramble II). Cells were harvested after 3 days and either lysed for RNA extraction to confirm Penk depletion or re-plated for assays of viable cell number (MTS assay), following treatment with 50 J/m² UV-C irradiation or 25 μM etoposide. (a) RT-PCR of RNA from 293 cells transfected with Scramble II, Penk 1 and Penk 2 siRNA confirmed depletion of Penk with siRNA. RNA isolated from 293 cells transfected with Scramble II, Penk 1 and Penk 2 siRNA was subjected to first strand synthesis using 1 μg of each RNA, and then reverse transcribed using oligo-(dT₁₅) and AMV reverse transcriptase. PCR was carried out using 25 ng of RNA (Penk, top left panel) and 1 ng (GAPDH, top right panel): densitometry of PCR bands was conducted to quantify PCR products (lower panel). (b) Whole-cell extracts of untreated 293 cells and 293 cells transfected with Scramble II, Penk 1 and Penk 2 siRNA were immunoblotted for Penk, using Penk antibodies (PE14 and PE19). Both Penk 1 and Penk 2 depleted a range of high-molecular-weight forms of Penk,¹³ although Penk 1 appeared to be more efficient. Actin immunoblot of same lysates confirmed equivalent protein loading. (c) Penk siRNA substantially preserves cell viability following UV irradiation. Change in cell viability in siRNA-transfected cells was measured in the MTS assay over a 72-h time course, following exposure to UV-C irradiation. Each data set was obtained from a representative experiment performed at least three times. Data points represent mean values (±S.E.M.) from wells in triplicate relative to control values (from non-irradiated, mock-transfected cells). (d) Penk siRNA delays cell death following exposure to the genotoxin etoposide. Change in cell viability over a time course following addition of etoposide was measured in the MTS assay, as above