Fig. S1: ΔNp63α is the predominant isoform in A431 and HaCaT cells. (A) quantitative
real time PCR (qRT-PCR) analysis of total cDNA from designated cell lines using
primers specific to TA or Np63 isoforms. H1299 lacking p63 was used as a negative
control. (B) Immunblot analysis using pan-p63 (4A4), Np63 specific (RR14), p63
isoform specific (H-129) and TAp63 specific antibodies and compared to overexpressed
p63 isoforms in H1299 cells. (C) TA and Np63 isoform specific qRT-PCR analysis of
HaCaT cells after treatment with non-silencing control (NSC) or a pan-p63 siRNA
showing equal knockdown of total p63 and both TAp63 and Np63 isoforms. Y-axis
represents relative fold change over H1299. * = p< 0.05.
Fig. S2: Np73 is the predominant p73 isoform in A431 and HaCaT cells. (A) qRT-PCR
analysis of total cDNA from designated cell lines using primers specific for the TA or
Np73 isoforms. (B) Immunblot analysis using pan-p73 or Np73 specific specific
antibodies and compared to p73 isoforms overexpressed in H1299 cells.
Fig. S3: Np63 is the predominant isoform responsible for changes in PTEN
expression. (A) A431 cells were transfected with non-silencing control (NSC) siRNA,
siRNA specific to the Np63 or TAp63 isoforms, or both. Total RNA was extracted and
transcript levels of TAp63, Np63 and PTEN were analyzed by qRT-PCR. Y-axis
represents the fold change in PTEN and p63 transcript levels relative to NSC transfected
cells. Immunoblots of p63, PTEN and -actin in A431 cells transfected as described
above are shown in the bottom panel. (B) HaCaT cells were transfected with nonsilencing control (NSC) siRNA, siRNA specific to Np63 or TAp73, or both. Total RNA
was extracted and transcript levels of TAp73, Np63 and PTEN were analyzed by qRTPCR. Y-axis represents the fold change transcript levels relative to NSC transfected
cells. * = p< 0.05. Immunoblots of p63, PTEN, and -actin in HaCaT cells transfected as
described above are shown in the bottom panel.
Fig. S4: p53 occupies the same locations of the pten promoter as p63. Chromatin
immunoprecipitation assays were performed on A431 and HaCaT cells wherein
chromatin was immunoprecipitated with normal IgG control antibody or an antibody
specific to p53 as indicated. Eluted DNA was PCR-amplified with primers specific for
multiple regions of the pten promoter. Specific nucleotide locations of the regions
amplified are shown in parentheses. The p21 promoter was used as a positive control,
however A431 cells harbor a mutation in p53 that affects DNA contact and may explain
why p53 fails to bind to the p21 promoter in these cells.
Fig. S5: Np63 suppresses nuclear PTEN. HaCaT cells were transfected with NSC or
p63 specific siRNA and subjected to indirect immunofluorescence for p63 and PTEN.
Nuclei are shown with DAPI staining. White arrows designate cells in which p63 was
silenced.