Figure 4

siRNA-mediated knockdown of Fau and Bcl-G expression attenuates UV-induced apoptosis in breast cancer cells. siRNA-mediated knockdown of Fau and Bcl-G expression, either alone or in combination, attenuates UV-induced apoptosis of T-47D breast cancer cells. T-47D cells were transfected with siRNA to Fau (FAU2), Bcl-G or negative control (NC). To observe the effects of combined knockdown, Fau plus Bcl-G siRNAs were co-transfected (Fau + Bcl-G); the controls for this group were cells transfected with double the amount of NC siRNA (2× NC). At 120 hours post transfection, samples were collected for the determination of Fau and Bcl-G transcript levels, and cells were either exposed to UV light (40 J/m²) or were mock-irradiated. (a) Real-time RT-PCR analysis of Fau (left-hand panel) and Bcl-G (right-hand panel) transcript levels. Data, expressed relative to the housekeeping gene ALAS1, are the mean ± standard error of the mean. *P < 0.05 versus NC siRNA, ^P < 0.05 versus 2× NC siRNA (Bonferroni's multiple comparison test); n = 5. (b) At 48 hours post UV exposure, the proportion of apoptotic cells (left-hand panel) was determined by a CaspaTag assay and fluorescence microscopy, and the short-term cell viability (right-hand panel) was determined by dye exclusion. Data for mock-irradiated (light bars) and UV-treated (dark bars) cells are the mean ± standard error of the mean. *P < 0.05 versus NC siRNA (UV-irradiated), ^P < 0.05 versus 2× NC siRNA (UV-irradiated) (Bonferroni's multiple comparison test); n = 5. Note that knockdown of either Fau or Bcl-G alone or in combination attenuates apoptosis induction by UV and that the extent of inhibition is similar for all three treatments. (c) Bcl-G transcript levels are reduced in ductal carcinoma of the breast. RNA was isolated from tumour and adjacent normal tissue (n = 18 patients), was reverse transcribed, and Bcl-G and ALAS1 transcript levels were determined using Taqman assays and real-time PCR using a relative standard curve protocol (cDNA from T-47D cells as standard). The Bcl-G/ALAS1 transcript ratio is reduced in tumour tissue. *P = 0.0021; paired Student's t test. Inset: scattergram of data.(d) Expression of Bcl-G was analysed in a cohort of 99 breast carcinoma patients and was correlated with survival data, as previously reported [27]. Kaplan–Meier survival curve showing no significant correlation between total Bcl-G expression levels and patient survival.