Figure 6
PHB2 knockdown inhibits 45S rRNA and 18S rRNA transcription and reduces occupancy of c-Myc on rDNA, but facilitates MyoD binding to rDNA in RD cells. (a) The 45S rRNA and 18S rRNA in RD cells were amplified by conventional SYBR real-time PCR analysis. After transfection, RNA extraction was performed. cDNA was prepared from total RNA using the TransScript First-Stand cDNA Synthesis SuperMix with random primers. Data are presented as the mean ± S.D. (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t test. (b) ChIP analysis of Myc binding to rDNA in RD cells transfected with siRNA. RD cells were transfected with PHB2 siRNA, collected and fixed after 48 h. Immunoprecipitated rDNA was quantitated by real-time PCR with two primer sets, H1 and H13. Data are presented as the mean ± S.D. (n = 3). (c) RD cells were prepared as in panel B, and then the ChIP assay of MyoD binding to rDNA was conducted. qPCR analysis of MyoD was conducted to check the quality of the ChIP-DNA, and five primer sets targeted to the rDNA E-box (H4, H8, H18, H27, H42) were utilized. Data are presented as the mean ± S.D. (n = 3).
