Fig. 1. SSRP1 enhances the transcriptional activity of p63γ and p73α, but not of p53 and ΔN-p63γ. (A) SSRP1 stimulates the transcriptional activity of p63 and p73, but not p53. H1299 cells were transfected with plasmids encoding p53 (50 ng), p63γ (50 ng) or p73α (50 ng) alone or with SSRP1 (750 ng) as indicated, along with reporters as described in Materials and methods. At 48 h post-transfection, cells were harvested for luciferase and β-gal assays. Luciferase activity was normalized by the internal β-gal activity and expressed in fold increase (each column represents the mean activity from three independent assays, and bars show standard deviation). (B) SSRP1, but not its N-terminal fragment, stimulates the p63γ-dependent luciferase activity. H1299 cells were transfected with pCDNA-Myc-p63γ (50 ng), pCDNA3-Flag-SSRP1 (1× 250 ng and 2× 500 ng) and pCDNA3-Flag-SSRP1/1–242 (500 ng) as indicated. Luciferase and β-gal assays were conducted as described above. Luciferase activity was expressed in arbitrary units here. (C) SSRP1 does not stimulate the ΔN-p63γ-dependent luciferase activity. The same transfection was carried out as that described above, except that pCDNA3-Myc-ΔN-p63γ (50 ng) was also used here in comparison with p63γ. A 50 ng aliquot of pCDNA3-Myc-p63γ and 500 ng of pCDNA3-Flag-SSRP1 were used in this experiment. The results shown in (A–C) were reproduced using p53 null H1299 cells and MEFs. (D) Western blot analysis of expression of exogenous p63γ and SSRP1. H1299 cells were transfected as described in the above panels, except that triplicate plates were used in each lane for detection of proteins. A 150 µg aliquot of proteins was loaded directly on an SDS gel. Flag-SSRP1 (F-SSRP1), Flag-SSRP1/1–242 (F-SSRP1/1–242) or Myc-p63γ was detected by western blot analyses using monoclonal anti-Flag or anti-Myc antibodies.

Fig. 2. SSRP1 stimulates the p63γ-dependent expression of endogenous p53 target genes. (A) RNA transcript analysis. H1299 cells (three 60 mm plates of cells for each lane) were transfected with 500 ng of pCDNA3-Myc-p63γ or pCDNA3-Myc-ΔN-p63γ alone or together with 2 µg of pCDNA3-Flag-SSRP1, as indicated on top. At 48 h post-transfection, cells were harvested for RNA preparation. RT–PCRs for six p53-responsive genes were conducted as described in Materials and methods and as indicated on the right. (B) Western blot analysis. The same transfection as described above was carried out except that only p63γ was used with 1 µg (1×) and 2 µg (2×) of SSRP1. Cells were harvested for preparation of cell lysates. Cell lysates containing 150 µg of proteins were loaded directly onto an SDS gel, followed by western blot analyses using antibodies as indicated on the right. (C) Cell cycle analysis of transfected p53 null MEFs as described above, except that GFP and GFP–p63γ were used.

Fig. 3. SSRP1 augments p63γ-induced apoptosis. (A and B) As indicated on the top of each panel, pCDNA3 plasmids encoding no protein as a control (V, 2 µg), p63γ (0.5 µg), ΔN-p63γ (0.5 µg) and/or SSRP1 (1 µg), together with the pGFP plasmid (200 ng), were introduced into 10⁵ H1299 cells/35 mm plate using LipofectAmine as described in Materials and methods. (A) At 32 h post-transfection, cells (200 in total) expressing GFP were counted under a fluorescence microscope using a blind approach, with morphologically round and shrunken cells (arrows) identified as apoptotic. (B) Images of total cells in the same views. (C) The percentage of apoptotic cells, with the standard error indicated by a bar above each column.

Fig. 4. SSRP1 interacts with p63γ and p73α, but not p53, in cells. H1299 cells (10⁶/60 mm plate, three plates each lane) were transfected with plasmids encoding no protein (3 µg), Myc-p63γ (1 µg), Ha-p73α (1 µg), p53 (1 µg) or Flag-SSRP1 (2 µg) either alone or together as indicated. At 30 h post-transfection, cells were harvested for preparation of cell lysates. A 300 µg aliquot of the lysates was used for immunoprecipitation with antibodies against Myc, Flag, p53 or p73, followed by western blotting with antibodies as indicated on the right. The results with the anti-Flag antibodies used for immunoprecipitation are shown in (A), while those with the anti-Myc antibodies are shown in (B). (C) The co-localization of GFP–p63γ with Flag-SSRP1 in the nucleus. Immunofluorescent staining of transfected H1299 cells was conducted as described in Materials and methods. Stained cells were examined under a deconvolution microscope. A representative image is shown here. The results of immunoprecipiation–western blot analysis for p73–SSRP1 interaction in cells is shown in (D) and (E), while that for p53–SSRP1 is shown in (F) and (G).

Fig. 5. SSRP1 binds directly to p63γ but not ΔN-p63γ in vitro. (A) GST fusion protein association assays were conducted as described in Materials and methods. A 300 ng aliquot of His-p63γ or His-ΔN-p63γ proteins was incubated with GST alone or GST–SSRP1 or deletion mutant fusion proteins that bound on the glutathione–Sepharose 12B beads (containing ∼1 µg of proteins). The GST fusion proteins used in this assay were stained with Coomassie Blue (the top panel). Bound p63 proteins were detected by western blotting using anti-p63 antibodies as shown in the two bottom panels. Thirty percent of ΔN-p63γ input was loaded directly onto the SDS gel as a reference. (B) The p63-binding fragment of SSRP1 impeded SSRP1-mediated stimulation of p63 activity. H1299 cells were transfected with plasmids encoding Myc-p63γ (50 ng) alone or with Flag-SSRP1 (500 ng) or Flag-250–490 (the p63-binding domain of SSRP1) (500 ng) along with reporters, and luciferase assays were carried out as described in Figure 1. (C) Western blot analysis of the levels of Myc-p63γ, Flag-SSRP1 and Flag-250–490 expressed in cells using antibodies against Myc and Flag, respectively. The asterisk denotes the degraded form of SSRP1 in lane 5.

Fig. 6. SSRP1 co-resides with p63γ at the p53RE sites of the endogenous MDM2 and p21^waf1 promoters. (A) ChIP analyses. H1299 cells were transfected with plasmids encoding no protein (3 µg), or with Myc-p63γ (1 µg) and/or Flag-SSRP1 (2 µg), as indicated on top. At 36 h post-transfection, cells were treated with a 1% formaldehyde solution for ChIP assays as described in Materials and methods. PCR products of 117 bp (top) encompassing the p53RE motif of the MDM2 promoter, 105 bp (middle) of the p21^waf1/cip1 promoter or 98 bp (bottom) of the non-p53RE sequence 2.5 kb upstream from the p53RE site of the MDM2 promoter were tested from the immunoprecipitates using antibodies against either Myc or Flag, as indicated on top. (B) Western blot analysis of the same transfected cells. Cell lysates containing 150 µg of proteins were loaded onto an SDS gel. Myc-p63γ and Flag-SSRP1 were detected with antibodies against Myc and Flag. (C) SSRP1 directly binds to the p53RE-bound p63γ. EMSAs were carried out as described in Materials and methods. In the reactions in lanes 1–9, 300 ng of poly(dIdC), 100 ng of wild-type (lane 2) or mutant (lane 3) p53RE oligomers, 500 ng of p63γ, 200 and 400 ng of SSRP1 (lanes 4 and 5), and 200 and 400 ng of BSA (lanes 6 and 7) were used as indicated on top. In the reactions in lanes 10–15, 400 ng of SSRP1 and 500 ng of anti-SSRP1 or anti-HA peptide antibodies were used in the presence or absence of 100 or 200 ng of poly(dIdC) as indicated on top.