FIGURE 7.

Phosphorylation of PHF8 promotes rRNA transcription. A, 293T cells were transfected with PHF8 siRNA or control siRNA and pHrD-IRES-Luc together with pRL-TK as an internal control. The cell lysates were harvested and subjected to Dual-Luciferase assay. B and C, 293T cells were transfected with pCMV FLAG-PHF8, pCMV FLAG-PHF8-S844A, or empty vector and pHrD-IRES-Luc together with pRL-TK as an internal control. The cell lysates were harvested and subjected to Dual-Luciferase assay (B). The pre-rRNA in transfected cells was quantified by RT-PCR, with GAPDH as an internal control (C). D–F, HeLa cells were transfected with pCMV FLAG-PHF8, pCMV FLAG-PHF8-S844A, or empty vector as a control. The cells were harvested and subjected to ChIP assays with primers for the rDNA promoter. D and E, immunoblot analysis was performed to check the expression of PHF8 and PHF8-S844A (F). G–I, 293T cells were transfected with CDK2 siRNA or control siRNA and rDNA luciferase reporter. The cell lysates were subjected to the luciferase assay (G). The total RNAs from transfected cells were extracted and used for real-time PCR for pre-rRNA (H). Immunoblot analysis was performed to check the expression of CDK2 (I). C, control. *, p < 0.05; **, p < 0.01.