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. 2014 Sep 9;289(43):29691–29700. doi: 10.1074/jbc.M114.571653

FIGURE 2.

FIGURE 2.

PHF2 inhibits rather than activates rDNA transcription. A, depletion of PHF2 and PHF8 by specific siRNAs. HeLa or HEK293T cells were transfected with siRNA against either PHF2 (siPHF2) or PHF8 (siPHF8) for 2 days, and the effect on the levels of endogenous PHF2 and PHF8 was determined by Western blot. Actin served as a loading control. siCtrl, nonspecific control siRNA. Concentration for each siRNA is 100 nm. B, Knockdown of PHF2 and PHF8 has opposite effects on rRNA transcription. HeLa or HEK293T cells were treated with control, siPHF2, or PHF8 as in A, and the effects on the levels of pre-rRNA transcripts were determined by quantitative RT-PCR analysis. Error bars, S.D.; n = 3. C, knockdown of PHF2 in HCT116 and A549 cells also led to increased rDNA transcription. HCT116 and A549 cells were transfected with siCtrl or siPHF2 for 2 days and then collected for Western blot analysis for PHF2 (left) or the levels of pre-rRNA transcripts by RT-PCR (right). The data are shown as levels of pre-rRNA transcription in siPHF2-treated cells relative to that of siCtrl-treated cells. Error bars, S.D.; n = 3. D, specific knockdown of PHF2 in HeLa or HEK293T cells by two distinct PHF2 shRNA constructs. HeLa or HEK293T cells were stably transfected with two different PHF2 shRNAs, ShPHF2-1 and ShPHF2-2, followed by Western blot analyses with antibodies against PHF2, PHF8, and β-actin. shCtrl, the control shRNA vector. E, knockdown of PHF2 with either of two distinct shRNAs led to increased expression of pre-rRNA. The relative levels of pre-rRNA in the HeLa and HEK293T cells stably transfected with shPHF2-1, shPHF2-2, or control shRNA were measured by qRT-PCR. Error bars, S.D.; n = 3. F, simultaneous knockdown of PHF2 and PHF8 nearly restored the normal level of rDNA transcription. HeLa cells were transfected with siCtrl, siPHF2, siPHF8, and siPHF2 + siPHF8, respectively, and the effect on the levels of PHF2 and PHF8 proteins was revealed by Western blot analysis (left), and the effect on rDNA transcription was determined by qRT-PCR analysis (right). Error bars, S.D.; n = 3.