Figure 11.

PA28γ-siRNA reduces SF2/ASF accumulation at transcription sites. 2E11 cells were cotransfected with pcDNA₃-Tat, pEGFP-SF2/ASF, and control- or PA28γ-siRNA (1) duplexes. After 48 h, cells were fixed and subjected to in situ hybridization using a MS2-Cy3 probe. (A) Right, merged images of the intracellular distribution of SF2/ASF-GFP (green) and MS2 TS and mRNA (red) under the indicated conditions. Left, magnification of the TS (white square): MS2 detection (red), SF2/ASF-GFP (green), merge (100× objective). Bar, 10 μm. (B) Quantification of the level of SF2/ASF-GFP recruited at the TS. The relative levels of SF2/ASF-GFP at the TS and at the NS was estimated by quantification of the green fluorescence using the Metamorph software, in cells expressing similar levels of transcripts (estimated by quantification of MS2-Cy3 at TS) and SF2/ASF-GFP in the NS. The values correspond to the means of four independent experiments (n = 40 cells, ±SD). See Figure S3 for illustration of the quantification procedure. (C) PA28γ-siRNA does not affect the level of transcription. Relative mRNA levels for the ribosomal S26 subunit (control) and for the artificial gene in 2E11 cells untreated or treated with the indicated siRNA were analyzed by RT-PCR and agarose gel electrophoresis.