Figure 4
OCT4 stimulates p63 but inhibits p53 expression. (a) Immunoblots of whole-cell lysates from 293FT cells 48 h after transfection. (b) The mRNA levels of p53 or p63 from 293FT cells at 48 h after transfection. Error bars presented as mean+S.D. from three independent experiments. **P<0.01. (c) Immunoblots of nuclear extracts (NE) from 293FT cells at 48 h after transfection with HA-OCT4 plasmid (WT) or a mutant (M): the start codon replaced with the stop codon. (d) p63 EMSA. P1, 2 or 3 represents probe 1, 2 or 3 containing site 1, 2 or 3 in the promoters. Left panel: lane 1, 4 or 7, probe alone; lane 2, 5 or 8, NE without HA-OCT4; and lane 3, 6 or 9, NE with HA-OCT4. Right panel: lane 1, probe alone; lane 2, NE without HA-OCT4; lane 3, NE with HA-OCT4; lane 4, added with HA anti-serum; lane 5, added with non-specific rabbit serum; and lane 6, added with cold probe. (e) p63 luciferase assay. The luciferase reporter containing the potential binding site, wild type (WT) or the different mutations that deleted site 1 (M1), 2 (M2) or 3 (M3) of the p63 promoter region. Error bars presented as mean+S.D. from three independent experiments. (f) p53 EMSA was similar to the description in panel (d). (g) p53 luciferase assay was similar to the description in panel (e) with wild type (WT) or different deleted mutation (M1 or M2) sequences of p53 promoter regions as described in Supplementary Figure S4c. (h) Data from a ChIP assay. Left panel: the DNA signals of the PCR product with the specific primers amplified from the cross-linked mixture (containing protein and Nt) immunoprecipitated (IP) with a HA antibody (for 293FT cells expressed with HA (293FT with HA) or HA-OCT4 (293FT with HA-OCT4)) or an OCT4 antibody (for H9 cells) as described in the Supplementary Information. M: marker. Right panel: quantification of the PCR products from the ChIP assay. Data shown are the average from two independent experiments
