The Rockefeller University Press $30.00
J. Cell Biol. Vol. 201 No. 3 427–437
Correspondence to Yohannes Tesfaigzi: firstname.lastname@example.org
Abbreviations used in this paper: Ad, adenovirus; BH, Bcl-2 homology; ChIP,
chromatin immunoprecipitation; CMV, cytomegalovirus; Ct, threshold cycle;
HAEC, human airway epithelial cell; HDAC, histone deacetylase; HDACi,
HDAC inhibitor; MAEC, murine airway epithelial cell; MEF, mouse embryonic
fibroblast; mTOR, mammalian target of rapamycin; PRD, proline-rich domain;
qRT-PCR, quantitative RT-PCR; shBmf, short hairpin Bmf; TSA, trichostatin A.
As a pleiotropic cytokine, IFN- mediates many of its antiviral
and anticancer properties directly by activating STAT1 (Hu and
Ivashkiv, 2009). The anticancer property of IFN- stems from
its ability to increase the susceptibility of many cell types, in-
cluding melanoma and colorectal cells, to undergo apoptosis in
response to cytotoxic chemotherapies (Borden et al., 2007).
IFN- induces apoptosis in not only carcinoma (Ossina et al.,
1997; Ruiz-Ruiz et al., 2000) but also primary cells (Trautmann
et al., 2000; Tesfaigzi et al., 2002a) by activating various path-
ways in a cell type–specific manner. In asthma, IFN- reduces
epithelial cell hyperplasia (Shi et al., 2002) by inducing expres-
sion of the BH3-only protein, Bik (Mebratu et al., 2008), and by
translocating Bax to the ER (Stout et al., 2007). We and others
have shown that IFN-–induced cell death is p53 independent
(Deiss et al., 1995; Ossina et al., 1997; Mebratu et al., 2008).
However, the effect of IFN- on p53 and the resulting cellular
conditions have not been reported.
The Bcl-2 family of proteins is characterized by the Bcl-2
homology (BH) domains. The prosurvival proteins, Bcl-2, Bcl-xL,
and Mcl-1, have four BH domains (BH1–4). The first group of
proapoptotic proteins, Bax, Bak, and Bok, is characterized by three
BH domains (BH1–3), whereas the second group contains only
the BH3 domain and, therefore, is designated as the BH3-only
group of proteins (Strasser, 2005). One of the BH3-only proteins,
Bmf, was first reported to cause cell death upon loss of cell at-
tachment (anoikis) by being released from dynein light chain
and inhibiting the function of the prosurvival Bcl-2 (Puthalakath
et al., 2001). Although Bmf was dispensable for anoikis in certain
cell types (Labi et al., 2008), it appears to play an essential role
for anoikis in others (Hausmann et al., 2011). In addition, anoi-
kis in human endothelial cells seem to involve Bmf (Schmelzle
et al., 2007), whereas those isolated from mouse (Labi et al., 2008)
do not. Because we have observed that airway epithelial cells
detach from the basement membrane during IFN-–induced reso-
lution of airway epithelial cells (Tesfaigzi, 2006), we investigated
this affects autophagy have not been reported. The present
study demonstrates that IFN- down-regulated expression
of the BH3 domain-only protein, Bmf, in human and mouse
airway epithelial cells in a p53-dependent manner. p53
also suppressed Bmf expression in response to other cell
death–stimulating agents, including ultraviolet radiation
and histone deacetylase inhibitors. IFN- did not affect
nterferon (IFN-)–induced cell death is mediated by
the BH3-only domain protein, Bik, in a p53-independent
manner. However, the effect of IFN- on p53 and how
Bmf messenger RNA half-life but increased nuclear p53
levels and the interaction of p53 with the Bmf promoter.
IFN-–induced interaction of HDAC1 and p53 resulted in
the deacetylation of p53 and suppression of Bmf expression
independent of p53’s proline-rich domain. Suppression of
Bmf facilitated IFN-–induced autophagy by reducing the
interaction of Beclin-1 and Bcl-2. Furthermore, autophagy
was prominent in cultured bmf/ but not in bmf+/+ cells.
Collectively, these observations show that deacetylation of
p53 suppresses Bmf expression and facilitates autophagy.
Deacetylation of p53 induces autophagy
by suppressing Bmf expression
Amelia U. Contreras,1 Yohannes Mebratu,1 Monica Delgado,1 Gilbert Montano,1 Chien-an A. Hu,2 Stefan W. Ryter,3
Augustine M.K. Choi,3 Yuting Lin,4 Jialing Xiang,4 Hitendra Chand,1 and Yohannes Tesfaigzi1
1Chronic Obstructive Pulmonary Disease Program, Lovelace Respiratory Research Institute, Albuquerque, NM 87108
2Department of Biochemistry and Molecular Biology, The University of New Mexico School of Medicine, Albuquerque, NM 87131
3Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
4Illinois Institute of Technology, Chicago, IL 60616
© 2013 Contreras et al. This article is distributed under the terms of an Attribution–
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as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
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JCB • VOLUME 201 • NUMBER 3 • 2013 428
HAECs (Fig. 1 B) and MAECs (Fig. 1 C). Treatment of bik/
(Fig. 1 D) or STAT1/ (Fig. 1 E) with IFN- also reduced Bmf
mRNA levels, suggesting that Bmf down-regulation was not
mediated by STAT1 or Bik. IFN- reduced all three isoforms of
Bmf mRNAs, BmfL, BmfCUG, and BmfS (Fig. 1 F). Similar to a
previous study for the thymus (Labi et al., 2008), we also de-
tected two Bmf proteins in the murine lung tissue representing
BmfCUG (25 kD) and BmfS (20 kD), which were absent in tissues
from bmf/ mice (Fig. 1 G); however, in contrast to the thymus,
BmfCUG was the predominantly expressed isoform in mouse lung
tissue (Fig. 1, G and I). Consistent with what was observed for
the Bmf mRNA isoforms, IFN- also reduced protein levels for
both BmfCUG and BmfS (Fig. 1 H). Because we did not expect a
proapoptotic protein to be reduced during IFN-–induced cell
death, we generated adenoviral expression vectors to investigate
whether one of the Bmf isoforms may inhibit cell death in airway
epithelial cells. We introduced a Kozak consensus sequence up-
stream of the BmfCUG transcriptional start site to allow expression.
Adenoviral expression of BmfCUG and BmfS showed that BmfCUG
appeared to be processed to BmfS, and the expressed proteins
were of the same size as the isoforms induced in MAECs by the
HDACi trichostatin A (TSA). However, both BmfCUG and BmfS
killed 50% of cells within 18 h after infection compared with
adenovirus (Ad)-GFP–infected controls (Fig. 1 I), demonstrat-
ing that expression of both isoforms is proapoptotic.
To investigate this paradox that the proapoptotic Bmf
was down-regulated by the cell death–inducing IFN-, we ex-
plored the Bmf mRNA levels in epithelial cancer cell lines, as
previous studies had described Bmf expression in various can-
cers (Puthalakath et al., 2001; Schmelzle et al., 2007). We noticed
that the p53-deficient cell lines, SOAS-2 and Calu-6 cells, showed
significantly higher Bmf mRNA levels compared with the p53-
sufficient cells, A549 and AALEB cells (Fig. 2 A). The role of
p53 in affecting Bmf expression was validated by expressing
p53 in Calu-6 cells using an adenoviral overexpression system
(Fig. 2 B). Interestingly, p53 expression alone was not sufficient,
but additional treatment with 10 mJ UV radiation, which is known
the effect of IFN- on Bmf expression in airway epithelial cells.
Bmf mRNA is transcribed from three start sites, but only two
isoforms, BmfCUG and BmfS, have been detected in murine thy-
mus, whereas the third isoform, BmfL, has not been detected so
far in any tissue (Grespi et al., 2010). In humans, BmfL is likely
not relevant because of a frame shift in the start site (Grespi
et al., 2010).
Histone deacetylase (HDAC) inhibitors (HDACis) induce
Bmf expression in a broad range of cancer cells by hyperacety-
lating histone tails (H3 and H4) at the Bmf promoter and facili-
tating transcription (Zhang et al., 2006a). Loss of Bmf protein
renders lymphocytes resistant to glucocorticoid- or HDACi-
induced cell death (Labi et al., 2008). HDACs were initially
known to target histones; however, it is now clear that many
other nonhistone proteins are substrates for various HDACs.
The first nonhistone protein known to be regulated by acetyla-
tion and deacetylation was p53 (Gu and Roeder, 1997; Tang
et al., 2006). Acetylation of human p53 at lysine 382 or murine
p53 at lysine 379 and acetylation in general have been shown to
be important for p53 stability, sequence-specific DNA-binding
activities, and recruitment of transcriptional activators (Itahana
et al., 2009). In the present study, we found that IFN- causes
deacetylation and nuclear accumulation of p53 to promote its
interaction with the Bmf promoter and suppress Bmf expression
and thereby facilitate autophagy.
Our previous studies demonstrate that physiologically relevant
levels of IFN- induce cell death in proliferating primary human
airway epithelial cells (HAECs), murine airway epithelial cells
(MAECs), and in AALEB cells, a cell line derived from HAECs
(Tesfaigzi et al., 2002b; Stout et al., 2007) by STAT1-dependent
Bik expression (Mebratu et al., 2008). Although screening for
the effect of IFN- on the expression of BH3-only proteins, we
found that IFN- reduced Bmf mRNA levels in AALEB cells
by fivefold (Fig. 1 A), and this reduction was replicated in both
Figure 1. IFN- down-regulates expression of proapoptotic Bmf isoforms. (A–E) Bmf mRNA levels in AALEB cells (A), HAECs (B), wild-type MAECs (C),
Bik/ MAECs (D), or STAT1/ MAECs (E) 48 h after treatment with 50 ng/ml IFN- as quantified by qRT-PCR. The relative standard curve method was
used for analysis of unknown samples, and data are presented as fold change after averaging the Ct values for the nontreated (NT) samples. (F) BmfS,
BmfCUG, and BmfL mRNA levels in IFN-–treated or untreated MAECs as analyzed by RT-PCR with GAPDH levels as controls. (G) Western blot of protein
lysates extracted from homogenized lung or thymus tissue from bmf/ and bmf+/+ mice and analyzed for Bmf expression. (H) Bmf protein levels in protein
lysates prepared from IFN-–treated or nontreated MAECs. (I) Bmf protein levels in protein lysates from MAECs treated with 300 nM TSA, infected with 100
MOI of Ad-BmfS, Ad-BmfCUG, or nontreated controls, and the percentage of viable cells. Cells were harvested and quantified using trypan blue exclusion.
Data presented are means ± SEM for three independent experiments. *, P < 0.05.
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429p53 suppresses Bmf to induce autophagy • Contreras et al.
half-lives among cells with and without p53 and IFN- treat-
ment suggested that p53 does not affect Bmf mRNA stability.
Inhibition of HDACs (HDACis) with sodium butyrate,
TSA, or MS-275 significantly increased Bmf mRNA in AALEB
cells (Fig. 3 C) and BmfCUG and BmfS protein levels in MAECs
(Fig. 1 I). Although Bmf mRNA levels were increased by TSA
in both p53/ and p53+/+ MAECs, Bmf mRNA expression was
twofold higher in p53/ compared with p53+/+ MAECs (Fig. 3 D),
suggesting that the inhibitory effect of p53 was still present in
cells treated with HDACis.
A previous study has established that overexpression of
HDAC1 suppresses Bmf expression by inhibiting the promoter
activity (Zhang et al., 2006a). Therefore, we next evaluated
whether IFN- affects the interaction of p53 with the Bmf pro-
moter. The Bmf promoter region at 97 is the region where
histone hyperacetylation occurs in cancer cell lines in response
to HDACi treatment to increase Bmf transcription (Zhang et al.,
2006a). We found by chromatin immunoprecipitation (ChIP)
that region 97, but not 560, of the Bmf promoter interacted
with HDAC1 in p53/ but not in p53+/+ HCT116 cells, and
IFN- had no effect on this interaction (Fig. 4 A). In AALEB
cells, p53 interaction with the Bmf promoter was absent in non-
treated controls but was present at region 97 and not 560
when cells were treated with IFN- (Fig. 4 B). These findings
suggest that HDAC1 requires the absence of p53 for its inter-
action with the Bmf promoter and that IFN- enhances the inter-
action of p53 with the Bmf promoter to suppress transcription.
Because the nuclear or cytosolic localization defines
p53 function (Lee and Gu, 2010), we assessed p53 localization
in IFN-–treated cells and found that compared with non-
treated controls, IFN- increased nuclear p53 levels, whereas
cytosolic p53 levels remained low and unchanged (Fig. 4 C).
Immunofluorescence also demonstrated that the percentage
of cells with nuclear p53 was significantly increased by IFN-
treatment (Fig. 4 D).
to activate p53 (Zhang and Xiong, 2001), was necessary for
reducing Bmf levels, whereas the same treatment significantly
increased Bmf mRNA levels in cells infected with adenoviral
GFP as a control (Fig. 2 C). Identical results were obtained
when p53 was expressed in Calu-6 cells using a lentiviral ex-
pression system after treatment with UV radiation (unpublished
data). Exposure of MAECs to 10 mJ UV radiation increased
p53 levels (Fig. 2 C) and reduced Bmf mRNA levels to 50% of
nontreated controls in both MAECs (Fig. 2 C) and AALEB cells
(Fig. 2 D) 6 h after treatment.
These findings suggested that suppression of Bmf expres-
sion by IFN- may be mediated by p53. Therefore, we investi-
gated the effect of IFN- on primary MAECs from p53/ and
p53+/+ mice and found that Bmf mRNA levels were signifi-
cantly up-regulated by IFN- in p53/ MAECs (Fig. 3 A), and
BmfCUG and BmfS proteins were detected in p53/ MAECs
treated with IFN- (Fig. 3 B). Similarly, suppression of p53
levels in AALEB cells using shRNA resulted in IFN- failing
to suppress Bmf mRNA levels (unpublished data). Having estab-
lished that p53 mediates IFN-–induced suppression of Bmf
expression, we investigated the possibility that p53 may be af-
fecting Bmf mRNA stability. In general, 3UTRs have conserved
sequences that regulate mRNA stability (Stoecklin and Anderson,
2007), and Bmf mRNA has an unusually long 4-kb 3UTR.
AALEB cells treated with and without IFN- were harvested
at 0, 0.5, 1, 2, and 4 h after blocking transcription with DRB
(5,6-dichloro-1--d-ribobenzimidazole). On average, the half-life
of Bmf mRNA in nontreated p53-sufficient A549 and AALEB
cells was 1.94 and 2.43 h and in the p53-deficient Calu-6 and
SAOS-2 cells, was 2.36 and 1.73 h, respectively. Bmf mRNA
half-life in IFN-–treated p53-sufficient and p53-deficient cells
was 1.5 and 1.8 h, respectively. The similarity of the mRNA
Figure 2. p53 mediates IFN-–induced Bmf suppression. (A) Bmf
expression levels in p53-sufficient and -deficient cell lines evaluated by
qRT-PCR. Bmf mRNA levels relative to p53-sufficient A549 are shown for
AALEB cells and the p53-deficient SAOS-2 and Calu-6 cells. Data pre-
sented are representative of four independent experiments. (B) Western
blot analysis of p53 in Calu-6 cells infected with an adenoviral expres-
sion vector for p53 and the relative Bmf mRNA levels quantified by
qRT-PCR in these cell lines when exposed to 10 mJ UV radiation com-
pared with the respective nontreated controls. (C) p53 protein levels in
MAEC 6 h after exposure to 10 mJ UV radiation and Bmf mRNA levels
quantified by qRT-PCR in MAECs exposed to10 mJ UV and nontreated
(NT) controls. (D) Bmf mRNA levels in AALEB cells 6, 12, and 24 h after
exposure to 10 mJ UV radiation compared with nontreated controls.
Data presented are means ± SEM for three independent experiments.
*, P < 0.05.
Figure 3. p53 suppresses IFN-– and HDACi-induced Bmf expression.
(A) Bmf mRNA in IFN-–treated and nontreated (NT) p53/ MAECs.
(B) Bmf protein levels in protein lysates prepared from p53/ MAECs
treated with nothing or IFN- for 48 h. (C) Bmf mRNA levels in AALEB
cells treated with 5 mM sodium butyrate, 300 nM TSA, and 5 µM MS-275
for 18 h compared with nontreated controls. (D) Bmf mRNA expression in
p53/ and p53+/+ MAECs treated with 300 nM TSA for 18 h. Error bars
indicate ±SEM (n = 3 independent experiments). *, P < 0.05; statistically
significant difference from controls.
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JCB • VOLUME 201 • NUMBER 3 • 2013 430
To determine whether prevention of p53 deacetylation
would abrogate the down-regulation of Bmf, we treated airway
epithelial cells with IFN- for 24 h followed by TSA in the
presence or absence of IFN- for an additional 24 h. Bmf was
up-regulated in the TSA + IFN-–treated compared with IFN-–
only treated control samples, suggesting that TSA prevented the
HDAC1 and p53 interaction (Fig. 5 C), further supporting the
idea that acetylation of p53 affects Bmf expression. Increased
accumulation of nuclear p53 and enhanced deacetylation of p53
in IFN-–treated cells was verified by double staining of non-
treated, IFN-–, TSA-, and TSA + IFN-–treated AALEB cells
(Fig. 5 D). These findings were further validated in immuno-
stained wild-type and AXXA mutant mouse embryonic fibro-
blasts (MEFs), and quantification showed that the percentage
of p53-positive cells was increased, whereas the percentage of
acetyl-p53–positive cells was decreased in IFN-–treated com-
pared with nontreated controls (Fig. 5 E). TSA increased the
acetylated p53 in control cells and in IFN-–stimulated cells in
both wild-type and AXXA mutant MEFs.
IFN-–induced deacetylation of p53 (Fig. 6 A) and sup-
pression of Bmf mRNA (Fig. 6 B) occurs in MAECs as early as
0.5 h after treatment, suggesting that it is a direct effect rather
than by other mediators that result from IFN- treatment. This
deacetylation of p53 was accompanied with the down-regulation
of Bmf mRNA. Furthermore, IFN- suppressed Bmf in p53-
sufficient wild-type colon epithelial cells (HCT116 p53+/+) but
increased Bmf mRNA levels in HCT116 p53/ cells within 0.5 h
after treatment (Fig. 6 C).
Although IFN- increased the levels of the proapoptotic
BmfCUG and BmfS in p53/ cells, cell death and the viability
of p53/ and p53+/+ MAECs remained unchanged (Fig. 6 D).
These results were confirmed in IFN-–treated AALEB cells,
In humans, the proline-rich domain (PRD) of p53 is de-
fined by residues 58–98, which contains 15 prolines and five
repeats of the amino acid motif PXXP (in which P designates
proline and X designates any amino acid). The histone acetyl-
transferase p300 binds to PXXP-containing peptides derived
from the proline repeat domain, and the PXXP motif in p53
is required for p53 acetylation by the transcription coactivator
p300 (Dornan et al., 2003). Therefore, we investigated whether
the PRD region plays a role in the IFN-–induced Bmf suppres-
sion. In mice, this PRD consists of two PXXP motifs, and we
obtained mice with a deletion of the PRD (p53P) or lacking the
four critical proline residues at loci 79, 82, 84, and 87 that make
up the tandem PXXP sites (p53AXXA; Toledo et al., 2006, 2007).
MAECs isolated from p53P, p53AXXA, and wild-type littermates
showed a reduction in Bmf levels when treated with IFN- simi-
lar to that observed in wild-type MAECs (Fig. 4 E).
p53 interacts with HDAC1 as part of a deacetylation
complex (Luo et al., 2000, 2004), and our experiments showed
that HDAC1 interacts with the Bmf promoter, and IFN- drives
the interaction of p53 with the Bmf promoter. Therefore, we in-
vestigated the interaction of p53 and HDAC1 in IFN-–treated
cells using immunoprecipitation assays and found that IFN-
increased HDAC1 levels in pull-down products using anti-
p53 antibodies (Fig. 5 A), demonstrating that IFN- en-
hanced the p53–HDAC1 interaction. Therefore, we assessed
the acetylation state of p53 after IFN- treatment and found
significantly reduced acetylated p53 levels in IFN-–treated
compared with nontreated cells (Fig. 5 A). An antibody spe-
cific to Lys382 was selected for detection because this is the site
mostly acetylated within p53 (Gu and Roeder, 1997). Deacety-
lation of p53 by IFN- was also observed in IFN-–treated
MAECs (Fig. 5 B).
Figure 4. p53 is enriched in the nucleus and interacts with the Bmf promoter. (A) ChIP assays on p53+/+ or p53/ HCT116 cells using a polyclonal anti-
body to HDAC1 or rabbit IgG1 as a control. DNA identification was performed using PCR with primers specific for Bmf promoter regions at 97 or 560
regions. (B) ChIP assay on AALEB cells nontreated (NT) or treated with IFN- for 48 h using a monoclonal antibody to p53 or mouse IgG1 as a control
using primers specific for Bmf promoter regions at 97 or 560 regions. Densitometry of PCR products from six independent experiments for DNA pulled
down with p53 normalized to IgG1 and IFN-–treated cells normalized to the nontreated values. IN, input. (C) Increased p53 protein levels in the nuclear
fractions of AALEB cells 48 h after treatment with IFN- compared with nontreated controls. Nuclear and cytosolic extracts were analyzed for p53, lamin,
and actin. Panel is representative of three independent experiments. (D) Representative photomicrographs of nontreated and IFN-–treated AALEB cells
immunostained with the anti-p53 antibody and quantification of p53-positive nuclei (n = 4/group). Bar, 15 µm. (E) Bmf mRNA levels in MAECs isolated
from p53P, p53AXXA, and p53wt littermates 48 h after IFN- treatment compared with nontreated MAECs (n = 3 independent experiments). WT, wild type.
Error bars indicate ±SEM. *, P < 0.05.
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431p53 suppresses Bmf to induce autophagy • Contreras et al.
IFN- induces autophagy in HeLa cells (Inbal et al., 2002)
and in gastric epithelial cells (Tu et al., 2011). We had also
observed that IFN- treatment increased both Beclin-1 and
conversion of LC3-I to LC3-II in AALEB cells and wild-type
MAECs (Fig. 7, A and B). Because the proapoptotic Bmf iso-
forms were suppressed by IFN-, we reasoned that Bmf may
have a role in suppressing autophagy and that IFN-–induced
down-regulation of Bmf may facilitate autophagy. Infection of
AALEB cells with Ad-BmfCUG to reconstitute the suppression
by IFN- or with Ad-GFP as a control showed that increased
Bmf expression reduced the levels of IFN-–induced Beclin-1
in which p53 was suppressed using shRNA targeting p53
(Fig. 6 E). Because Bcl-2 blocks the proapoptotic function
of Bmf (Puthalakath et al., 2001), we tested whether IFN- in-
creases expression of antiapoptotic proteins, Bcl-2 and Bcl-xL,
and found that IFN- increased Bcl-2 and Bcl-xL protein lev-
els in p53/, but not in p53+/+, MAECs (Fig. 6 F). Although
these experiments suggest that increased expression of these
antiapoptotic proteins may suppress apoptosis in IFN-–treated
p53-deficient MAECs when BmfCUG and BmfS are increased,
the role of IFN- suppressing the proapoptotic Bmf was still
Figure 5. IFN- causes HDAC1–p53 interaction and deacetylates p53. (A) HDAC1 interacts with p53. Nuclear lysates were prepared from AALEB cells
after treatment with IFN- for 48 h and were immunoprecipitated (IP) using a p53-specific monoclonal antibody. The nuclear lysates (input) and immuno-
precipitates were resolved by SDS-PAGE and analyzed using Western blotting with antibodies to HDAC1, total p53, and acetyl-p53. Panel is representative of
three independent experiments. (B) Nuclear extracts prepared from IFN-–treated and nontreated MAECs and probed for total p53, acetyl-p53, and lamin.
(C) Bmf mRNA levels in IFN-–, TSA-, or IFN- + TSA–treated AALEB cells compared with nontreated (NT) controls. (D) Representative photomicrographs
IFN-–, TSA-, or IFN-/TSA-treated or nontreated AALEB cells immunostained for p53 (shown in red) and acetyl-p53 (shown in green). Nuclei were stained
with DAPI (shown in blue), and merged images are shown in the right-most column. Bars, 10 µm. (E) Quantification of the percentage of cells positive for
p53 and acetyl-p53 (Ac-p53) in wild-type (WT) and AXXA MEFs either nontreated or treated with IFN-, TSA, or IFN-/TSA. Error bars show group means ±
SEM (n = 4/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 6. IFN- deacetylates p53 but does not affect IFN-–induced cell death. (A) Nuclear extracts from MAECs 0.5 h after IFN- treatment or nontreated
(NT) controls that were probed for total p53, acetyl-p53, and lamin. (B) Bmf mRNA levels in p53+/+ HCT116 cells 0.5 h after IFN- treatment or nontreated
controls. *, P < 0.05. (C) Bmf mRNA in p53/ and p53+/+ MAECs treated with IFN- for 0.5 h (n = 3 independent experiments). (D) Quantification of
viable p53/ and p53+/+ MAECs treated with IFN- for 48 h as assessed by trypan blue exclusion. (E) Quantification of viable AALEB cells transfected
with an empty vector plasmid (shRNA control [CTR]) or one expressing p53 shRNA were treated with IFN- for 48 h. (F) Bcl-xL and Bcl-2 protein levels in
protein lysates prepared from p53/ and p53+/+ MAECs treated with nothing or IFN- for 48 h. Error bars indicate ±SEM.
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JCB • VOLUME 201 • NUMBER 3 • 2013 432
process. Although the number of mitochondria between bmf/
and bmf-+/+ MAECs showed no difference, there appeared to
be morphological differences with more damaged mitochon-
dria being present in bmf/ compared with bmf-+/+ MAECs.
To determine the physiological relevance of reduced Bmf expres-
sion causing autophagy, we exposed bmf-+/+ and bmf/ MEFs
to autophagy-promoting conditions and found that starvation
(Fig. 7 H) and treatment with pp242 (Fig. 7 I), a mammalian
target of rapamycin (mTOR) inhibitor, caused enhanced cell
death in bmf/ compared with bmf-+/+ MEFs. These findings
suggest that increased baseline autophagy in bmf/ MEFs
reduces the protective role of autophagy in these cells. Suppres-
sion of Bmf in p53/ HCT116 cells using short hairpin Bmf
(shBmf) increased autophagy (Fig. 7 J), and IFN-–induced
and the processing of LC3-I to LC3-II (Fig. 7 C). Although
lung or thymus tissues from bmf/ showed no evidence of in-
creased Beclin or LC3-II compared with those from bmf-+/+
mice (Fig. 7 D), cultured but nontreated bmf/ MAECs and
MEFs showed increased levels of Beclin-1 and accumulation
of LC3-II compared with bmf-+/+ MAECs (Fig. 7 E). The for-
mation of autophagosomes in cultured MAECs and MEFs was
also indicated by transfection of pmCherry-LC3, displaying
significantly increased punctation in bmf/ compared with
bmf-+/+ MAECs and MEFs (Fig. 7 F). In addition, transmission
electron micrographs revealed significantly more autophagic
vacuoles per unit area in bmf/ compared with bmf-+/+ MAECs
(Fig. 7 G). Some cells displayed mitochondria surrounded by
membranous material that may be evidence for a premitophagic
Figure 7. IFN- suppresses Bmf expression to induce autophagy. (A and B) IFN- increases protein levels of Beclin-1 and LC3B in AALEB cells (A) and
MAECs (B) compared with nontreated (NT) controls as detected by Western blot analysis. (C) AALEB cells treated with IFN- for 48 h and infected with
50 MOI of Ad-BmfCUG or Ad-GFP. Bmf expression inhibits IFN-–induced Beclin-1 and LC3B protein levels. (D) Western blot of lung and thymus tissues from
bmf+/+ (wild type [WT]) and bmf/ (knockout [KO]) mice probed for Beclin-1 and LC3B proteins. (E) Western blot analysis of protein extracts from bmf+/+
(wild type) and bmf/ (knockout) MAECs and MEFs probed for Beclin-1, LC3B, and -actin. (F) Representative micrographs of bmf+/+ and bmf/ MAECs
and MEFs expressing mCherry-LC3B and cells with punctuate LC3B were quantified from >50 bmf+/+ and bmf/ MAECs. (G) Representative electron
micrographs of bmf+/+ and bmf/ MAECs that were cultured in 6-well dishes. Thin sections of cells were analyzed by scanning electron microscopy. The
arrowhead denotes an autophagic vesicle, and the arrow denotes a mitochondrion surrounded by a double membrane. Quantification of autophagic
vesicles per 100 µm2 in >30 each bmf+/+ and bmf/ MAECs. (H) Quantification of viable bmf+/+ and bmf/ MEFs maintained in starvation media for
4 or 24 h relative to cell grown in regular media (n = 3 independent experiments). (I) Quantification of viable bmf+/+ and bmf/ MEFs 24 h after treatment
with the mTOR inhibitor, pp242, at 2.5 µM relative to nontreated control cells (n = 3 independent experiments). (J) Knockdown of Bmf mRNA using shBmf
in p53/ HCT116 cells reduced Bmf mRNA levels. Western blot analysis of shRNA control (shCtr)– and shBmf-transfected p53/ HCT116 cells probed
with Beclin-1, LC3B, and -actin antibodies. The Western blot is representative of three experiments using three different shBmf constructs. (K) Western blot
of IFN-–treated p53+/+ and p53/ HCT116 cells probed with Beclin-1, LC3B, and -actin antibodies. (L) Immunoprecipitation of protein extracts from
IFN-–treated and nontreated bmf+/+ and nontreated bmf/ MEFs using anti–Beclin-1 and Western blot analysis of input and immunoprecipitates (IP) with
Bcl-2, Beclin-1, Bmf, and -actin antibodies. Results are representative of four independent immunoprecipitations. Error bars indicate ±SEM. *, P < 0.05;
**, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
on April 30, 2013
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433p53 suppresses Bmf to induce autophagy • Contreras et al.
colon adenocarcinoma cells (Ossina et al., 1997), and gastric
cancer cells (Gao et al., 2010), have shown that IFN-–induced
cell death is p53 independent. However, IFN- may require
p53 in certain cell types, as p53 accumulation is associated
with IFN- sensitizing hepatocytes to apoptosis induced by
genotoxic stress in mice that overexpress IFN- in the liver
(Lüth et al., 2011).
IFN-–induced p53 deacetylation was accompanied with
nuclear accumulation of p53. Therefore, it is possible that nu-
clear export mechanisms for p53 involve acetylation. Although
the PXXP motif in p53 is required for p53 acetylation by the
transcription coactivator p300 (Dornan et al., 2003), our find-
ings show that this motif appears to be dispensable for the
deacetylation. p53 is acetylated on the eight lysine residues in
the DNA-binding and C-terminal regions, and such acetyla-
tion prevents interaction with Mdm2 and leads to stabiliza-
tion and increase in the p53 protein (Tang et al., 2008). The
classical importin-– pathway is responsible for import of
only nonubiquitinated p53 into the nucleus during the early
stages of stress response, such as DNA damage (Marchenko
et al., 2010). Future studies will investigate whether lysine
residues other than Lys382 are deacetylated by IFN- and
whether this modification minimizes the interaction of p53
with nuclear export proteins, such as MDM2-mediated ubiq-
uitination (Boyd et al., 2000), which may result in increased
nuclear p53 levels.
Although the importance of p53 acetylation on transcrip-
tional activation has been studied, the functional significance
of deacetylated p53 remains unknown. Our findings suggest
that p53 blocks the interaction of HDAC1 with the Bmf pro-
moter and that deacetylated p53 is likely to be part of a repres-
sive complex responsible for the down-regulation of Bmf. Many
other genes have been reported to be suppressed by p53, includ-
ing DNA topoisomerase II, cyclin B, Cdc2 (Yun et al., 1999),
MMP-1 and -13 (Sun et al., 2000), presenilin-1 (Roperch et al.,
1998), myc, and Map-4 (Kidokoro et al., 2008). In general, tran-
scriptional activation requires p53 to bind a consensus sequence
(Wei et al., 2006); however, the repression mechanism by p53 is
not well studied. For example, p53 directly binds to the Mad1L1
promoter, but no p53 consensus site was found (Chun and Jin,
2003). Similarly, we found that p53 interacts with the Bmf pro-
moter in IFN-–treated cells, although the bmf upstream region
lacks a consensus p53 response element. Although DNA bind-
ing is required for p53 to suppress cdc2 (Yun et al., 1999) or
cdc20 (Banerjee et al., 2009), p53 can also suppress by interfer-
ing with transactivating factors (St Clair et al., 2004). Future
studies will elucidate whether similar mechanisms are involved
for p53 to suppress Bmf expression.
HDACis consistently increased Bmf expression in pri-
mary HAECs and MAECs, and p53 suppressed the extent of
Bmf induction. The fact that p53 dampens HDACi-induced
Bmf expression supports the idea that deacetylated p53 inter-
acts with the Bmf promoter to suppress its activity. Similar to
our findings, the HDACis induce acetylation of histones H3 and
H4 at the Bmf promoter region in various human cancer cell
lines, and ectopic expression of HDAC1 reduces Bmf expres-
sion (Zhang et al., 2006a). The previous studies also showed
autophagy was increased in p53+/+ but reduced in p53/ HCT116
cells (Fig. 7 K), suggesting that IFN-–induced autophagy is
p53 dependent. To investigate the mechanism by which Bmf
may be regulating autophagy, we assessed whether Beclin-1–
Bcl-2 interaction is affected by the presence or absence of Bmf.
Although Bcl-2 levels are higher in bmf-+/+ compared with
bmf/ MEFs the difference is more evident after immuno-
precipitation with Beclin-1 antibodies. Beclin-1–Bcl-2 interaction
was also reduced when bmf-+/+ MEFs were treated with IFN-
consistent with the observation that IFN- reduces Bmf levels
and thereby enhances autophagy (Fig. 7 L). These findings sug-
gest that the Beclin-1–Bcl-2 interaction is stabilized by the pres-
ence of Bmf, and loss or even reduced levels of Bmf releases
Beclin-1 to cause autophagy. Bmf was not detected in the input
but only in the immunoprecipitate from nontreated bmf-+/+ MEFs
because it was concentrated by the immunoprecipitation pro-
cess. Together, these findings suggest that IFN-, by deacetyl-
ating p53, suppresses Bmf expression to facilitate autophagy.
The present experiments show that IFN- induces autophagy
by suppressing Bmf expression through HDAC1-mediated
deacetylation of p53. A novel finding of this study is that IFN-
caused deacetylation of human p53 at Lys382 and murine p53
at Lys379. We were compelled to investigate the role of IFN- in
affecting p53 acetylation because HDAC1 interacts with the Bmf
promoter to suppress its activity, and IFN- modified the inter-
action of p53 with the Bmf promoter but had no effect on Bmf
mRNA half-life. These findings suggest that p53 and HDAC1
affect promoter activity by affecting chromatin remodeling. Our
finding is consistent with a previous study that deacetylation of
p53 is mediated by an HDAC1-containing complex (Murphy
et al., 1999). IFN-–induced deacetylation of p53 and suppres-
sion of Bmf mRNA occur as early as 0.5 h after IFN- treat-
ment, suggesting that these events are a direct effect of IFN-.
However, the IFN-–induced signaling pathways that lead to
HDAC1–p53 interaction and whether other proteins are involved
in deacetylating p53 remain to be clarified. SIRT1 (Sirtuin 1),
a mammalian NAD+-dependent HDAC, also deacetylates p53
and may be involved in this process (Liu et al., 2011).
The fact that IFN- significantly decreased the number of
AALEB cells that are immunopositive for acetylated p53 sug-
gests that acetylated p53 present in nontreated cells may have a
role for the regular proliferation of HAECs and MAECs. Loss
of acetylation completely abolishes p53-dependent growth ar-
rest and apoptosis (Tang et al., 2008) by abrogating the sequence-
specific transcriptional activity of p53 (Gu and Roeder, 1997;
Luo et al., 2000). These studies are consistent with our findings
that IFN-–induced cell death utilizes a pathway that is inde-
pendent of p53. The observation that p53+/+ and p53/ MAECs
are equally susceptible to IFN-–induced cell death is sup-
ported by our previous study demonstrating that IFN-–induced
Bik, the central mediator of IFN-–induced cell death, causes
cell death in a p53-independent manner (Mebratu et al., 2008).
Similarly, other studies in various cell types, including breast car-
cinoma and neuroblastoma cell lines (Porta et al., 2005), human
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Published April 29, 2013
JCB • VOLUME 201 • NUMBER 3 • 2013 434
Materials and methods
Pathogen-free STAT1/, p53+/ mice were purchased from The Jackson
Laboratory, and bik/ and bmf/ mice on the C57BL/6 background
were previously described (Coultas et al., 2004; Labi et al., 2008) and
were made available by A. Strasser (Walter and Eliza Hall Institute,
Parkville, Australia). Mice with modified PRD p53P and p53AXXA (Toledo
et al., 2007), on the 129J background, were obtained from G.M. Wahl
(The Salk Institute for Biological Studies, La Jolla, CA). These mice along
with the wild-type littermates were bred at the Lovelace Respiratory Research
Institute under specific pathogen-free conditions and genotyped as described
previously (Labi et al., 2008). STAT1/, p53+/ mice were genotyped
using protocols provided by The Jackson Laboratory. All animal experi-
ments were approved by the Institutional Animal Care and Use Committee
and at the Lovelace Respiratory Research Institute, a facility approved by
the Association for the Assessment and Accreditation for Laboratory Animal
The preparation of MAECs was performed as described previously (You
et al., 2002). In brief, tracheas were cut open lengthwise and incubated
in pronase solution (DMEM, 1.4 mg/ml pronase, and 0.1 mg/ml DNase)
overnight at 4°C. Enzymatic activity was stopped with 10% FBS (Invitro-
gen), and cells were collected by gently rocking tracheas in DMEM/Ham’s
H12 media (Invitrogen) followed by centrifugation at 400 g for 10 min
at 4°C. Cells were incubated in 5 ml of declumping solution (DMEM
and 2 mM EDTA) and plated on collagen-coated plates. The immortalized
HAECs, AALEB cells (Stout et al., 2007), and HAECs (Takara Bio Inc.) were
maintained in bronchial epithelial growth medium (Lonza). The p53-deficient
SAOS-2 and Calu-6 and the p53-sufficient small airway carcinoma-derived
A549 cell lines were maintained in bronchial epithelial growth medium
with penicillin/streptomycin, FBS, and l-glutamine. HCT116 p53/ and
p53+/+ cells (a gift from B. Vogelstein, Johns Hopkins University, Baltimore,
MD) were maintained in McCoy’s medium supplemented with FBS. MEFs
from bmf+/+ and bmf/ mice were cultured in DMEM supplemented with
10% FBS and were used for experiments at passages 3–15. We used Earle’s
Balanced Salt Solution (Sigma-Aldrich) as a starvation medium. Cells were
seeded on 6-well tissue-culture plates and cultured to 60–70% confluency
before irradiation with 10 mJ UV light using a UV cross-linker (Stratalinker
1800; Agilent Technologies).
Quantitative RT-PCR (qRT-PCR)
Extraction of RNA from cell pellets was performed using the RNeasy kit
(QIAGEN), and concentration was determined using a spectrophotom-
eter (NanoDrop 1000; Thermo Fisher Scientific). The primer/probe sets for
Bmf, CDKN1B, and 18s were obtained from Applied Biosystems. Target
mRNAs were amplified by quantitative real-time PCR in 20-µl reactions
on the real-time PCR system (PRISM 7900HT; Applied Biosystems) using
the One-Step RT-PCR Master Mix (TaqMan; Applied Biosystems). Relative
quantities from duplicate amplifications were calculated by normalizing
averaged threshold cycle (Ct) values to CDKN1B and/or 18s to obtain Ct,
and the relative standard curve method was used for determining the fold
change as described previously (Schwalm et al., 2008). For qRT-PCR, prim-
ers specific for mouse GAPDH, 5-AGGCCGGTGCTGAGTATGTC-3 and
5-TGCCTGCTTCACCACCTTCT-3, were used for normalizing RNA levels.
Immunoprecipitation and Western blot analysis
Total protein lysates or cytosolic and nuclear fractions were prepared
(Mebratu et al., 2008), and protein was analyzed by Western blotting as
previously described (Stout et al., 2007). In brief, cells were lysed in NP-40
to obtain the cytosolic fraction, and nuclear fractions were prepared
extracting the nuclear pellet with a hypertonic extraction buffer (50 mM
Hepes, pH 7.8, 50 mM KCl, and 300 mM NaCl) in the presence of prote-
ase and phosphatase inhibitors. For immunoprecipitation using the cross-
link immunoprecipitation kit (Thermo Fisher Scientific), cells were rinsed
twice with cold PBS, scraped into cold PBS plus protease inhibitors, and
analyzed per the manufacturer’s instructions. We used the rat anti-Bmf
monoclonal antibody, a gift from A. Strasser and A. Villunger (Innsbruck
Medical University, Innsbruck, Austria) at 2 µg/ml, and the rabbit anti-p53
polyclonal antibody (FL-393; Santa Cruz Biotechnology, Inc.), acetyl-p53
(Lys382; Cell Signaling Technology), rabbit antilamin polyclonal (#2032; Cell
Signaling Technology), or rabbit anti-HDAC1 polyclonal (EMD Millipore)
were used at a 1:1,000 dilution. We tested various Bcl-2 antibodies, in-
cluding those obtained from BD (catalog nos. 554279 and 554087) and
that overexpression of histone acetyltransferase p300 mimics
the effects of the HDACis, suggesting that Bmf expression in
cancer cells is primarily regulated by histone hyperacetylation
(Zhang et al., 2006b), and this approach was the basis to pro-
mote HDACis for cancer therapy. However, the present study
shows that HDACis also induce Bmf in primary airway epithe-
lial cells, suggesting that the use of these compounds for cancer
therapy may be associated with side effects and should be ap-
proached with caution.
Although the role IFN- plays in cell death has been es-
tablished, the role of IFN- in inducing autophagy in airway
epithelial cells and the mechanisms involved have not been
previously reported. IFN- increases Beclin-1 expression and
the conversion of LC3I to LC3II, suggesting the formation of
autophagosomes. We find that IFN-–induced suppression of
Bmf plays a critical role in allowing autophagy to proceed be-
cause restoring Bmf expression suppressed the induction of
Beclin-1 and the processing of LC3I to LC3II. However, in
p53/ cells in which IFN- induces Bmf expression, autoph-
agy was suppressed. The observation that there is no evidence
for autophagy in the lung or thymus tissues from bmf/ mice,
but autophagy is enhanced in cultured bmf/ MAECs and
MEFs, suggests that Bmf is crucial in suppressing autophagy
in cells when there is minimal stress, such as present in cul-
ture conditions. The fact that increased autophagy in bmf/
MEFs reduces the protective role of autophagy in these cells
supports the overall idea that IFN-, by reducing Bmf expres-
sion, sensitizes cells to death. Grespi et al. (2010) used MEFs
from wild-type and knockout mice that were immortalized with
SV40 and tested the effect of serum deprivation or inhibition
of the phosphatidylinositol-3-kinase/AKT/mTOR network over
time, and death was reduced by Bmf deficiency in these cells.
It is possible that starvation media cause autophagy that
is different from FCS withdrawal or mTOR inhibition, or the
response may be different in primary MEFs. Although IFN-–
induced autophagy in melanoma cells (Yan et al., 2011) and
in gastric cells (Tu et al., 2011) has recently been reported,
the mechanisms by which this autophagy is mediated are un-
known. Our experiments suggest that IFN-, by reducing the
interaction of Beclin-1 with Bcl-2, enhances autophagy and
that Bmf stabilizes the Beclin-1–Bcl-2 interaction because loss
of Bmf diminished the interaction of Beclin-1 and Bcl-2. To
our knowledge, this is the first study to show that Bmf is a
potent inhibitor of autophagy. A previous study showed that
Ras-induced expression of Noxa and Beclin-1 promotes auto-
phagic cell death by Noxa displacing Bcl-2 from Beclin-1
(Elgendy et al., 2011). In contrast, Bim inhibits autophagy by
recruiting Beclin-1 to microtubules (Luo et al., 2012). To-
gether, these findings add a new dimension to the role of the
BH3-only protein family in regulating autophagy. p53 plays a
dual role as a positive and a negative regulator of autophagy
(Maiuri et al., 2010). Nuclear p53 is reported to encourage
autophagy through transcriptional control of specific genes,
whereas cytoplasmic p53 has an inhibitory effect (Tasdemir
et al., 2008). Our findings suggest that suppression of Bmf by
nuclear p53 may be one of the mechanisms by which autoph-
agy is enhanced by nuclear p53.
on April 30, 2013
Published April 29, 2013
p53 suppresses Bmf to induce autophagy • Contreras et al.
was confirmed with PCR using the following primers specific for the Bmf
promoter: region 560, 5-ACCTAAGGGCTCCCCTGGA-3 and 5-GCA-
GGTCGGAAGAAAACTGCAGC-3, and region 97, 5-TTGGCGCTTC-
ACTCGCCATT-3 and 5-ATCCCGCAAACAGCTGAT-3.
Transmission electron microscopy
Cells used for electron microscopy analysis were fixed in 2.5% glutaral-
dehyde and postfixed with 1% osmium tetroxide before being dehydrated
in ethanol. Tissues were infiltrated with propylene oxide and embedded
in Agar 100 resin for preparation of ultrathin sections. After staining with
uranyl acetate and lead citrate, sections were examined using a transmis-
sion electron microscope (JEM 1210; JEOL) at 80 or 60 kV onto electron
microscope film (ESTAR Thick Base; Kodak). Electron micrographs (n = 30)
for each sample were quantified for mitochondria, and autophagosome
number and relative area were calculated using ImageJ software (National
Institutes of Health).
Fold changes or scanned density values were averaged and compared for
significance between groups using the Student’s t test. Data were analyzed
using Prism statistical analysis software (GraphPad Software), and P < 0.05
was considered statistically significant.
The authors thank Elizabeth Bennett and Kurt Schwalm for technical assistance
on selected experiments. We thank Louise Trakimas of the Harvard Electron
Microscopy Core for electron microscopy histology and imaging.
These studies were supported by grants from the National Institutes of
Health (HL068111 and ES015482) and by the Flight Attendant Medical
Submitted: 10 May 2012
Accepted: 29 March 2013
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p53 downregulates Cdc20 by direct binding to its promoter causing
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Santa Cruz Biotechnology, Inc. (sc-492, sc-7382, and sc-578), and found
that sc-7382 and sc-578 showed a single 26-kD band representing Bcl-2.
Therefore, the rat anti–Bcl-2 monoclonal sc-578 was used for immunopre-
cipitation and Western blot experiments. The Bcl-2 and the rabbit anti–
Bcl-xL polyclonal (sc-634; Santa Cruz Biotechnology, Inc.) antibodies were
used at 1:300 and 1:200 dilutions, respectively. For immunoprecipitation
experiments, rabbit anti–Beclin-1 (Abcam) was used, but for Western blot-
ting, rabbit anti–Beclin-1 and rabbit anti-LC3B (Cell Signaling Technology)
antibodies were both used at 1:1,000. The mouse actin polyclonal anti-
body was used at 1:5,000 (Santa Cruz Biotechnology, Inc.), and goat
anti–rat, goat anti–rabbit secondary, or rabbit anti–mouse antibodies were
used for visualizing proteins with chemiluminescence (PerkinElmer) using
the image reader (LAS-4000; Fujifilm).
For immunofluorescence, AALEB cells grown on eight-chamber slides
(Lab-Tek II; Thermo Fisher Scientific) were fixed using 3% paraformalde-
hyde with 3% sucrose in PBS and permeabilized using 0.2% Triton X-100
with 0.2% saponin in a blocking solution containing 3% IgG-free BSA, 1%
gelatin, and 2% normal donkey serum. Cells were probed with anti-p53
(#sc126; clone DO1; Santa Cruz Biotechnology, Inc.), anti–acetyl-p53
(Lys382; #2525; Cell Signaling Technology), or isotype controls at a 1:
500 dilution. MEFs were probed with anti-p53 (#sc6243; clone FL393;
Santa Cruz Biotechnology, Inc.) or antiacetyl-p53 (Lys379; #2570; Cell
Signaling Technology). The immunolabeled cells were detected using F(ab)2
fragments of respective secondary antibodies conjugated to Dylight-549
or -649 (Jackson ImmunoResearch Laboratories, Inc.) at a 1:500 dilution
and mounted with Fluoromount-G (SouthernBiotech) containing DAPI for
nuclear staining. Fluorescently labeled cells were analyzed as described
previously (Stout et al., 2007) using an imaging system (Axioplan 2; Carl
Zeiss) equipped with a charge-coupled device camera (ORCA-ER; Hama-
matsu Photonics) coupled with a wavelength switch (Lambda DG-4; Sutter
Instrument) and acquisition software (SlideBook 5; Intelligent Imaging In-
novations, Inc.). Quantification of p53-positive or acetyl-p53–positive cells
was conducted by counting ≥400 cells/well by a person unaware of the
Adenoviral and retroviral expression vectors
Adenoviral expression vectors for p53 and BmfCUG and BmfS were de-
veloped by cloning the respective cDNAs into the shuttle vector. RNA
was isolated from MAECs, and 1.5 µg was converted to cDNA using the
cDNA synthesis kit (SuperScript; Invitrogen). RT-PCR was performed using
the following primers: BmfS, 5-AAAATGGAGCCACCTCAGTGTGTG-3
and 5-TCACCAGGGCCCCACCCCTTC-3; BmfCUG, 5-AAACTGGCG-
CAGAGCCCTGGCATCA-3 and 5-TCACCAGGGCCCCACCCCTTC-3;
and BmfL, 5-ATGCCCGGAGCGGGCGTATTT-3 and 5-TCACCAG-
GGCCCCACCCCTTC-3. Shuttle cytomegalovirus (CMV)_p53, shuttle
CMV_BmfCUG, and shuttle CMV_BmfS were packaged into virus-producing
cells, and Ad particles were harvested by collecting media at the time when
cells were rounding up as described previously (Xiang et al., 1996).
The retroviral silencing vector encoding for p53 shRNA and the
control vector were purchased from OriGene. Retroviral vectors expressing
the respective shRNA or control constructs were packaged in Phoenix by
transfecting using FuGENE (Promega), and the harvested virus was used to
infect AALEB cells as previously described (Mebratu et al., 2008).
Bmf mRNA half-life
Cells were treated with the RNA polymerase inhibitor DRB (Sigma-Aldrich)
at a final concentration of 50 ng/ml for SAOS-2, Calu-3, A549, and
Calu-6 and at 100 ng/ml for AALEBs to stop RNA polymerase activity. The
relative mRNA abundance was calculated using the Ct method, and
mRNA half-life was calculated using the Greenberg formula by averaging
the calculated slope from mRNA levels over time (Greenberg, 1972).
After treating with 50 ng/ml IFN- for 48 h, cells were fixed with 1% form-
aldehyde, and the reaction was quenched using 1.25 M glycine and
scraped into cold PBS containing protease inhibitors. ChIP was performed
using the Magna ChIP G kit (MAGNA0002; EMD Millipore) as described
by the manufacturer. Sonicated nuclear fractions were incubated with
mouse IgG1 monoclonal antibody to p53 (sc-98 Pab1801; Santa Cruz
Biotechnology, Inc.), rabbit polyclonal antibody to HDAC1 (06–720; EMD
Millipore), rabbit polyclonal antibody to acetyl-H4 (06–598; EMD Millipore),
rabbit polyclonal antibody to acetyl-H3 (06-599B; EMD Millipore), mouse
IgG1, or rabbit as a control (CBL600; EMD Millipore). DNA identification
on April 30, 2013
Published April 29, 2013
JCB • VOLUME 201 • NUMBER 3 • 2013 436
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