1. PUMA, a Novel Proapoptotic Gene, Is Induced by p53
Katsunori Nakano, Karen H Vousden 
Molecular Cell 
Volume 7, Issue 3, Pages (March 2001)
DOI: /S (01)

2. Figure 1 Identification of PUMA
(A) The genomic structure of PUMA showing the exon–intron organization and the alternative transcripts. Transcripts 1, 2, and 3 were identified in this study; transcript 4 was identified by Yu et al. in the accompanying paper. The predicted initiation codons are also indicated; transcript 1 contains two alternative, overlapping open reading frames. Also shown are the two potential p53 binding sites in the first intron (PUMA BS1 and PUMA BS2) compared to the consensus p53 binding site (p53 CBS).
(B) The predicted amino acid sequence of PUMA-α, -β, -γ, and -δ. Sequences that are common between the proteins are shown in the same color, and the BH3 domain is underlined.
(C) The BH3 domain in PUMA-α and -β compared with other Bcl-2 family members
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2 PUMA Expression Is p53 Inducible
(A) Northern blot using an exon 4–specific probe, showing expression of PUMA in p53-inducible SAOS-2 and H1299 cells without (−) and with (dox) doxycycline treatment and IMR90 cells without (−) and with (Adr) adriamycin treatment. One predominant PUMA transcript of approximately 1.6–1.8 kb was detected. GAPDH expression was examined as a loading control.
(B) Expression of PUMA in RKO, RKO/E6, MCF-7, and U2OS cells treated with actinomycin D, detected as in (A).
(C) Northern blot showing activation of PUMA expression following activation of p53 in response to p14ARF expression in NARF cells, detected as in (A).
(D) Semiquantitative RT–PCR showing activation of expression of p53 and several p53-responsive genes (PUMA, p21WAF1/CIP1, PIG3, Bax, and p53R2) at various time points following doxycycline treatment of p53-inducible SAOS-2 cells.
(E) Northern blot showing that the PUMA transcript induced by p53 in RKO but not RKO/E6 cells is recognized by exon 1b–, exon 3–, and exon 4–specific probes.
(F) Semiquantitative RT–PCR showing elevated expression of PUMA transcripts 1, 2, and 3 in response to p53 activation in U2OS cells. The identity of each of the amplified bands was confirmed by sequencing
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3 Identification of a p53 Binding Site in PUMA Intron 1
(A) DNA binding activity of p53 to oligonucleotides containing two consensus p53 binding sites (PG13), PUMA binding site 1 (PUMA BS1), PUMA binding site 2 (PUMA BS2), and mutant PUMA binding site 2 (mt PUMA BS2). The DNA binding activity of in vitro–translated p53 protein was activated using the C-terminal anti-p53 antibody pAb421 as indicated.
(B) Competitive inhibition of p53 binding to the PUMA binding site 2 with the wild-type PUMA BS2 but not the mutant PUMA BS2 oligonucleotide
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4 Endogenous PUMA Expression Is Induced by p53
(A) PUMA-α and PUMA-β expression following transient transfection of U2OS cells. PUMA proteins were detected by Western blotting using the anti-PUMA rabbit antiserum generated in this study. Lysate from cells transfected with vector sequences only were used as the control.
(B) Detection of Flag-PUMA-β following transient transfection of U2OS cells. Lysates from PUMA-β- transfected or vector only–transfected cells were immunoprecipitated using the anti-PUMA antiserum, and coprecipitated proteins were detected by Western blotting using an anti-Flag antibody.
(C) Expression of PUMA-β in RKO cells either untreated (control) or treated with actinomycin D to activate p53 or LLnL to inhibit proteasome-mediated protein degradation, as indicated. PUMA-β was detected by immunoprecipitation followed by Western blotting using the anti-PUMA antiserum.
(D) Expression of PUMA-α in p53-inducible H1299 cells in the presence or absence of doxycycline. Mitochondrial fractions of the cells were Western blotted and probed with protein A–purified anti-PUMA antiserum.
(E) Expression of PUMA-β in cells treated as in (D). PUMA-β was detected using antigen-purified anti-PUMA antiserum
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5 Localization of PUMA
(A) Expression of Flag-tagged PUMA-α, PUMA-β, ΔLRR-PUMA-α, ΔLRR-PUMA-β (lacking the first three amino acids of the BH3 domain; Figure 1C), PUMA-γ, and PUMA-δ following transient transfection of U2OS cells.
(B) Immunofluorescent analysis of U2OS cells transiently transfected with Flag-tagged PUMA proteins as indicated. Localization of the PUMA proteins was determined using an anti-Flag antibody; location of the mitochondria was revealed using the mitochondrial marker CMXRos
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6 PUMA Inhibits Colony Formation and Induces Apoptosis
(A) U2OS and H1299 cells were transfected with plasmids expressing the indicated proteins and a neomycin resistance marker (pCMV is the control expressing only neomycin resistance). After transfection, the cells were selected for 14 days in G418-containing medium, then fixed and stained. Colony formation in representative dishes of transfected U2OS cells is shown. The same results were obtained in H1299 cells or using PUMA-α (data not shown).
(B) Expression of Flag-PUMA-α or Flag-PUMA-β in inducible H1299 cells following treatment with doxycycline for the indicated times. Protein expression was determined by Western blotting with an anti-Flag antibody.
(C) Flow cytometric analysis of H1299 cells following induction of PUMA-α or PUMA-β expression. Both PUMA-α and PUMA-β efficiently increase the apoptotic rate (as measured by cells with a sub-G1 DNA content) 24 hr after induction with doxycycline.
(D) Expression of Flag-PUMA-β in inducible SAOS-2 cells following treatment with doxycycline for the indicated times. Protein expression was determined by Western blotting with an anti-Flag antibody.
(E) Flow cytometric analysis of SAOS-2 cells following induction of PUMA-β expression for the indicated times
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7
Bcl-2 Binds PUMA-α and PUMA-β and Overcomes PUMA-Mediated Growth Arrest
(A) Bcl-2 protein was immunoprecipitated from U2OS cells following transient transfection with Bcl-2 and the indicated Flag-tagged PUMA expression constructs. The immunoprecipitated proteins were then Western blotted and probed for PUMA (anti-Flag) and Bcl-2 expression. Western blotting of the cell lysate before immunoprecipitation confirmed equivalent expression of each protein.
(B) PUMA-β and PUMA-δ were immunoprecipitated from U2OS cells following transient transfection with Flag-tagged PUMA and Bcl-2 expression constructs. The immunoprecipitated proteins were then Western blotted and probed for Bcl-2 and PUMA (anti-Flag) expression. Western blotting of the cell lysate before immunoprecipitation confirmed equivalent expression of each protein.
(C) Colony formation of U2OS cells following cotransfection of PUMA-β and Bcl-2 expression constructs as indicated. Twenty-four hours after transfection, the cells were split 1:10 and 1:2.5, as indicated, then drug selected for 14 days. The colonies were then fixed and stained
Molecular Cell 2001 7, DOI: ( /S (01) )

9. Figure 8
PUMA-α and PUMA-β Induce Cytochrome c Release and Activate Procaspases 3 and 9
(A) Immunofluorescence showing localization of cytochrome c and DNA (DAPI) in PUMA-α or PUMA-β inducible H1299 cells before (control) and after (dox) activation of PUMA expression by treatment with doxycycline. Apoptotic cells showing nuclear condensation and cytochrome c release from mitochrondrial stores are seen following PUMA activation. Cotreatment of the cells with the caspase inhibitor z-VAD-fmk inhibits apoptosis but does not prevent release of cytochrome c in response to PUMA expression.
(B) Western blot analysis of caspase 9 and caspase 3 following induction of PUMA-β by treatment of SAOS-2-PUMA-β cells with doxycycline for the indicated times. Cleavage of procaspase 9 and procaspase 3 to the smaller active forms indicates activation of this apoptotic pathway. β-actin expression was assessed to ensure equal protein loading
Molecular Cell 2001 7, DOI: ( /S (01) )

10. Figure 9 Inhibition of PUMA Expression Reduces p53-Mediated Apoptosis
p53-SAOS-2 cells were left untreated or were treated with 0.5 μg/ml doxycycline to induce p53 in the presence of either control or antisense PUMA oligonucleotides, as indicated. After 24 hr, the apoptotic rate was measured by flow cytometry, as described
Molecular Cell 2001 7, DOI: ( /S (01) )