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Fig. 6. Changes in GAS5 expression modulate the survival of human peripheral blood lymphocytes. Peripheral blood lymphocytes were stimulated with 2.5 µg/ml PHA and transfected with siRNAs targeting GAS5 or with negative control (–)siRNA. (A,B) Numbers of viable cells were determined by vital-dye staining, at the indicated time points (mean ± s.e.m. from seven independent experiments), either (A) in the presence of serum or (B) in its absence (mean ± s.e.m. from seven independent experiments). (C) Apoptosis was determined at different time points using CaspaTag (mean ± s.e.m. from five independent experiments). (D) The expression of endogenous GAS5 in lymphocytes transfected with GAS5-specific siRNA relative to cells transfected with negative control siRNA was determined 72 hours post transfection, by real-time RT-PCR (mean ± s.e.m. from seven separate experiments). (E) Peripheral blood lymphocytes were cultured in complete RPMI medium supplemented with PHA for 5 days and transiently transfected with pCMVSPORT only, or with pCMVSPORT containing GAS5 ESTs, using the Nucleofector (Amaxa; program T-23). After 4 hours, the medium was changed to medium that contains 2.5 µg/ml of PHA. The level of GAS5 overexpression was determined by real-time RT-PCR and was found to be four- to fivefold. Numbers of viable cells were determined by vital-dye staining. Data represent the mean ± s.e.m. from seven independent experiments. *P<0.01 compared with vector only. (F) Expression analysis of GAS5 splice variants by RT-PCR. RNA from PHA-stimulated (untransfected) primary lymphocytes was extracted at 24, 48, 72 and 96 hours. RNAs were reverse transcribed, and PCR was performed on the cDNA using GAS5-forward primer located in exon 9 and GAS5-reverse primer in exon 12. Three PCR fragments were observed. As verified by sequence analysis, all products correspond to GAS5 splice variants. (G) GAS5 expression in stimulated primary lymphocytes increases with time in culture. Expression of endogenous GAS5 in primary lymphocytes was determined by real-time PCR, using the comparative CT method, normalised with 18S RNA as an internal control. Results are represented as the mean ± s.e.m. from three separate experiments. *P<0.01 compared with (–)siRNA.
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