Suppression of histone mRNA transcription by knockdown of PHAX. (A) U2OS cells were transfected with the indicated siRNAs. After 48-h incubation, nascent RNAs were labeled by 5EU for 1.5 h. 5EU-labeled nascent mRNAs were determined by qRT-PCR analysis. (B–D) U2OS cells were transfected with siPHAX. After 48-h incubation, RNAPII-binding DNA levels on the indicated gene loci were determined by ChIP assay. (E) U2OS cells were cotransfected with siPHAX, an Rluc-expressing plasmid (pGL4-SV40p-Rluc), and the reporter plasmid carrying HIC, H2AX, or GAPDH promoter-driven Fluc expression cassette. After 48-h incubation, Fluc mRNA levels were determined by qRT-PCR analysis. Fluc mRNA levels were normalized by Rluc mRNA levels. Data are the means ± S.D. (n = 3–4). (*) P < 0.05, (**) P < 0.01.
