Fig. 6. Representative FDD (in gray scale) detecting p53-regulated changes in gene expression after tetracycline removal from DLD-1 or H1299 cells with inducible wild-type p53. Four RNA samples representing 9 and 12 h with Tet (left two lanes) and without Tet (right two lanes), respectively, were compared. Primer combinations detecting changes in gene expression are as indicated (e.g., G3 = G-anchor + HAP-3, etc.). Four of the clearly induced genes (G20, G54, G63, and G116) after sequencing turned out to be p53 itself, validating the comprehensiveness and precision of our FDD platform. The nature of other p53-induced genes is summarized in SI Table 1.

Fig. 7. Inducible expression of GFP-Killin leads to rapid termination in cell proliferation and apoptosis. (A) Cell proliferation rate for DLD-1 cells with tetracycline-regulated expression of either GFP-Killin or GFP alone was compared with or without induction, as indicated. Both attached (live) and detached (dying) cells were counted and combined for each time point, as indicated. (B) FACS analysis of cell-cycle profiles. The cells showed little decrease in S phase DNA content or increase in G₁ or G₂/M DNA content during the first 48 h of GFP-Killin induction, when growth arrest became apparent (see A). Massive apoptosis (subG1 content) was apparent after 3 days of induction. (C) Western blot analysis of PARP cleavage. Caspase-mediated cleavage of PARP was evident from day 2 before DNA fragmentation seen by FACS.

Fig. 8. RNAi knockdown of killin expression blocks p53-mediated apoptosis. FACS analysis of p53-3 cells stably transfected with either pSuper-killin or pSuper vector control confirmed that blocking p53-dependent killin expression would essentially prevent cells from apoptosis, without affecting G₁ arrest mediated by p21. The p53 induction time after tetracycline withdrawal was as indicated. Results are representative of multiple clones of cells in duplicated experiments.

Fig. 9. The full-length native Killin is a DNA-binding protein. In vitro transcribed and translated Killin (K) or vector alone (V) were labeled with 35S and incubated with either single-stranded (ss) or double-stranded (ds) DNA cellulose. After washing with PBS, bound proteins were resolved on a 15% SDS/PAGE gel and visualized by autoradiography. Killin (20 kDa) but not the nonspecific protein (100 kDa) from the vector alone was specifically retained by DNA cellulose.

Fig. 10. In vitro DNA-binding kinetics of Killin/N8-50 peptide. ³²-P-end-labeled double-stranded, single-stranded, and artificial replication fork DNA templates of 32-35 bases or base pairs in length were each incubated with increasing concentrations of Killin/N8-50 peptide as indicated. The reactions were resolved on a 6% TBE PAGE gel. The Killin-DNA-binding kinetics was quantified by counting the radioactivity of the complex formed (upper retained band) from each reaction in duplicate.

Fig. 11. Colocalization of RFP-Killin with S phase cells labeled by BrdU. Experiments were carried out as described in Fig. 5. (A) Two additional representative nuclei showing mutually exclusive nuclear patterns of RFP-Killin and BrdU-labeled replication forks. (B) Representative views of cell populations visualized simultaneously for RFP/BrdU and RFP-Killin/BrdU. (C) The predicted and observed percentages of cells with colocalization of either RFP/BrdU or RFP-Killin/BrdU were quantified below the respective images.

Fig. 12. Activation of DNA damage checkpoint control network by inducible GFP-Killin. Western blot analysis was carried out for Chk1and Chk2 and their phosphorylation status using phospho-specific antibodies as indicated. GFP-Killin (detected by GFP antibody) was induced in DLD-1 cells by tetracycline withdrawal, as indicated.