Mybbp1a negatively regulates the expression of ribosomal RNA. (A) Establishment of the control (ctrl) and Mybbp1a (si-Mybbp1a) knockdown HeLa stable lines. Expression of Mybbp1a was assessed by Western blot (left) or real-time RT-PCR (right) analysis. Actin serves as the internal control. (B) qRT-PCR analysis of pre-rRNA expression in control or Mybbp1a knockdown cells derived from proliferating (unsync), G1/S-arrested (G1/S), or mitotic (M) culture. (C) Control (ctrl) or Mybbp1a (si-Mybbp1a) knockdown cells were incubated in media containing the indicated glucose concentrations (4500, 1000, or 300 μg/ml) for 24 h. Total RNA was extracted from these cells and analyzed for pre-rRNA expression using qRT-PCR. (D) HeLa cells were transiently transfected with an empty vector (ctrl) or a construct encoding Myc-Mybbp1a. Expression of the ectopic Mybbp1a and actin (loading control) was examined by Western blot analysis. (E) & (F) Cells from (D) were cultured in either normal (E) or serum-free (F) media for 24 h, prior to qRT-PCR analysis of pre-rRNA expression. For bar graphs, data presented are normalized to GAPDH values, with the mean ± SD values from at least three experiments also shown (ns, not significant; *p < 0.05; **p < 0.01).