Skip to main content
. 2013 Mar;33(5):887–903. doi: 10.1128/MCB.00637-12

Fig 6.

Fig 6

The repression of DNMT1 contributes to HBP1-induced premature senescence and replicative senescence. (A) DNMT1 rescues HBP1-induced S-phase inhibition. 2BS cells (PD15) infected with pBabe, HBP1, or HBP1 and DNMT1 were labeled with BrdU and stained at day 14 after infection. The percentages of BrdU-positive nuclei (means ± standard errors of the means) were determined in three independent experiments (>300 cells counted per experiment). Statistical differences were analyzed using the t test; P < 0.05. (B) HBP1 is necessary for DNMT1 knockdown-induced S-phase inhibition. 2BS cells (PD30) infected with pSR, HBP1shRNA, DNMT1shRNA, or HBP1shRNA and DNMT1shRNA were labeled with BrdU and stained at day 14 after infection. The percentages of BrdU-positive nuclei (means ± standard errors of the means) were determined in three independent experiments (>300 cells counted per experiment). Statistical differences were analyzed using the t test; P < 0.05. (C) DNMT1 attenuates HBP1 increase in population doubling time. 2BS cells (PD15) were infected with pBabe, pHBP1, or pDNMT1 and PD measured in a time course experiment. Day 0 represents the 14th day postinfection. The means ± standard errors of the means for three independent experiments are shown. Statistical differences were analyzed using the t test; P < 0.05. (D) HBP1 and DNMT1 knockdown-induced increase in population doubling time. 2BS cells (PD30) were infected with pSR, pHBP1shRNA, pDNMT1shRNA, or pHBP1shRNA and pDNMT1shRNA. The experiment was performed as described for panel A. (E) DNMT1 prevents HBP1-induced SA β-Gal expression. 2BS cells (PD15) were infected with pBabe (as a control), HBP1, or HBP1 and DNMT1. Cells were then stained for SA β-Gal at day 14 after infection (top). The percentage of cells positive for SA β-Gal in 2BS cells infected with pBabe, HBP1, or HBP1 and DNMT1 was determined in three independent experiments and expressed as mean ± standard error of the mean, with >300 cells per experiment (bottom). Statistical differences were analyzed using the t test; P < 0.01. (F) HBP1 is required for DNMT1 knockdown-induced senescence. 2BS cells (PD30) were infected with pSR, HBP1shRNA, DNMT1shRNA, or HBP1shRNA and DNMT1shRNA. SA β-Gal expression was measured as described for panel C. (G) Abrogation of both HBP1 and DNMT1 abolishes senescence and promotes tumorigenesis. WI-38 cells were doubly infected with shRNAs to DNMT1 and to HBP1 and then selected for simultaneous decreases in both genes. The resulting cells were analyzed for transformation (growth in soft agar). About 500,000 HBP1KD-DNMT1KD cells were implanted subcutaneously in NOD-SCID mice, which were monitored for ∼6 weeks for tumor growth. (H) Suppression of DNMT1 by HBP1 participates in Ras-induced premature senescence. Left, DNMT1 eliminates the capability of Ras upregulating p16 expression. Levels of p16 and GAPDH (as a control) were determined by Western blotting of 2BS cells at PD15 infected with pBabe, Ras, or Ras and DNMT1. Right, DNMT1 prevents Ras-induced SA β-Gal expression. 2BS cells (PD15) were infected with pBabe, Ras, or Ras and DNMT1. Cells were then stained for SA β-Gal at day 14 after infection. The percentage of cells positive for SA β-Gal is shown. Statistical differences were analyzed using the t test; P < 0.01.