Figure 5

Effects of mutation of MSP58 NLSs on ribosomal gene transcription. A and B, pol I-dependent transcription of prHu3-Luc reporter (left). prHu3-Luc reporter plasmid (firefly) and the internal control plasmid pRL-TK (Renilla) were co-transfected into HT1080 or HeLa stable cell lines as indicated. After 36 h, both firefly and Renilla luciferase activities were measured. Columns, mean of three independent experiments; bars, S.D. *, p < 0.05. **, p < 0.01. NS, non-significant. Quantitative RT-PCR analysis of 47S rRNA HT1080 or HeLa stable cell lines (middle). Values for 47S rRNA were normalized to the GAPDH housekeeping control. The expression of 47S rRNA in vector control cells was defined as 1.0, with other values were accordingly normalized. Columns, mean of three independent experiments; bars, S.D. **, p < 0.01. ***, p < 0.001. NS, non-significant. (Right) HT1080 and HeLa cells were transfected with control or MSP58-knockdown (pSuper-MSP58 si-3) plasmid. At 48 h after transfection the expressions of 47S rRNA were analyzed by quantitative RT-PCR. GAPDH was amplified as a control. MSP58 protein levels were assessed by Western blotting. Columns, mean of three independent experiments; bars, S.D. **, p < 0.01. C. Schematic representation of the 47S gene organization and position of the specific primers used for ChIP assays (left). HT1080 cells stably expressing wild-type and NLS mutants (5A and 10A) MSP58 were fixed with formaldehyde, and cross-linked chromatin was precleared and immunoprecipitated with anti-HA antibody or normal IgG (negative control) as indicated. Eluted DNA was analyzed by a PCR using primers corresponding to the 47S gene promoter sequence, and the resultant DNA fragments of 344 bp were separated on 2% agarose gels. “Input” bands were obtained from DNA purified from chromatin not yet immunoprecipitated. Experiments were repeated three times.