Supplementary Materials
Supplementary Results
Ubiquitous BMCC1 expression in mouse and human tissues
A gene of BMCC1 encodes at least two long and short transcriptional variants,
BMCC1/PRUNE2-1 and PRUNE2/PRUNE2-2, respectively. PRUNE2 overlaps the
5′-end of the BMCC1 transcript (Supplementary Figure S1a). In order to investigate the
protein products of BMCC1 (340-kDa) and PRUNE2 (30-kDa), we employed two
antibodies raised against specific fragments of BMCC1 and PRUNE2 (Supplementary
Figure S1a). Specificity of the anti-BMCC1 antibody was validated by immunoblotting
and immunostaining (Supplementary Figure S1). Utilizing them, expression profiles of
BMCC1 and PRUNE2 were examined in the wild type mouse. BMCC1 was detected as
a 340-kDa band by either anti-BMCC1 or anti-PRUNE2 antibodies in a wide variety of
mouse organs. The anti-PRUNE2 antibody also detected a 30-kDa molecule in the same
samples (Supplementary Figure S2a).
This same pattern of BMCC1 expression was mirrored by immunohistochemical
analysis using anti-BMCC1 antibodies to probe human tissues (Supplementary Figure
S2b). For example, BMCC1 was detected in neuronal tissues derived from the cerebrum
and cerebellum as well as in muscle, adrenal gland, liver and pancreas. BMCC1
expression was markedly elevated in the epithelial cells of multiple organs. These
results are consistent with our previous finding that BMCC1 mRNA, monitored by
semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), is
expressed in various human tissues 16.
BMCC1, and not PRUNE2, is expressed in human NB and LNCaP cell lines
BMCC1 mRNA is expressed in NB, sarcoma and melanoma cell lines
16
, and not in
most non-NB human cell lines. Therefore, we performed immunoblotting analyses
using anti-BMCC1 and anti-PRUNE2 antibodies to determine BMCC1 and PRUNE2
expression levels in BMCC1-positive NB cell lines (Supplementary Figure S2c). The
highest expression of BMCC1 using either antibody was detected in LNCaP cells, a
prostate cancer cell line, as previously reported
44
. SK-N-AS, NBL-S, NGP and NB9
cells expressed moderate levels; NLF, SK-N-BE and SK-N-DZ cells expressed low
levels and the expression was undetectable in 293T cells. Because PRUNE2 was not
detectably expressed in any of these cell lines (Supplementary Figure S2c), we focused
on endogenous BMCC1 in the following study. In addition, we examined expressions of
TrkA and p53 among these cells.
Supplementary Figure Legends
Supplementary Figure S1 Specificity of antibodies raised against BMCC1 and
PRUNE2. (a) Functional motifs and domains in the primary structure of BMCC1
(PRUNE2-1) and PRUNE2 (PRUNE2-2). The black and red stars indicate epitopes
recognised by anti-PRUNE2 and anti-BMCC1 antibodies, respectively. (b) Comparison
of the amino acid residues of BH3 homology motifs in BCL2 and BNIP family proteins.
(c) Flag-tagged BMCC1 was overexpressed in 293T cells in which native BMCC1 was
not detected. Expression of Flag-BMCC1 was detected at 340-kDa by either
anti-BMCC1 (left panel) or anti-Flag antibody (right panel), respectively. (d)
Flag-BMCC1, Flag-PRUNE2 or empty vector was transfected in 293T cells. Indirect
immunofluorescence stain utilizing DAPI (blue), anti-BMCC1 antibody (green), and
anti-Flag antibody (red) are shown. (e) Specificity of anti-BMCC1 antibody was shown
by indirect immunostaining in BMCC1-positive prostate cancer cell line LNCaP. (f)
BMCC1-positive (LNCaP) and -negative (PC3) cells were employed as controls for
immunohistochemistry (IHC). Anti-BMCC1 antibody detects BMCC1 protein on IHC
(g).
Supplementary Figure S2
Expression of BMCC1 in normal tissues and human
cancer cell lines. (a) Expression profiles of BMCC1 and PRUNE2 in 18 types of mouse
tissues. Actin was used as a dose control. (b) IHC utilizing anti-BMCC1 antibody
(brown) and hematoxylin counterstain (purple) demonstrates BMCC1 expression in 20
types of human tissues. (c) Immunoblot demonstrates expression levels of BMCC1,
PRUNE2, TrkA, and p53 in human cancer cell lines. 293T cells in which Flag–tagged
PRUNE2 was transfected was employed as a control. The status of p53 gene was
indicated (wild type, -; mutated, +).
Supplementary Figure S3
BMCC1 decreased the level of phosphorylation at T308
in AKT and at S241 in PDK1. Full-length BMCC1 was overexpressed in HeLa (a), and
other cell lines (b). (c) Phosphorylation level of PDK1-S241 was largely enhanced by
shRNA-mediated knockdown of BMCC1 in NB cell lines, NBL-S and SK-N-AS cells.
Supplementary Figure S4
Subcellular localization of FOXO3a and accumulation of
BIM was promoted by BMCC1 harbouring BNIP2 homology region at the C-terminal.
(a) Translocation of FOXO3a into the nucleus was facilitated by full-length BMCC1,
and not BMCC1C, in HeLa (left panel) and NBL-S (right panel) cells. Indirect
immunofluorescent microscopy was performed at the time point indicated after
transfection. Positive cells regarding either GFP, Flag-BMCC1, or Flag-BMCC1C
were indicated by arrowheads. (b) Cellular accumulation of BIM was promoted by the
overexpression of full-length BMCC1 in HeLa cells. Immunostain was performed at 24
h after transfection.
Supplementary Figure S5
BMCC1 was associated with BCL2 in human cells. (a)
Interaction between endogenous BMCC1 and BCL2 was demonstrated by
immunoprecipitation utilizing an anti-BMCC1 antibody in LNCaP cells in which BCL2
was overexpressed. (b) Flag-tagged BMCC1 and endogenous BCL2 were
co-immunoprecipitated by an anti-Flag antibody in HeLa cells. (c) Subcellular
localization of BMCC1 and BCL2 in human cancer cell lines was shown in biochemical
fractions, such as whole cell lysate, cytoplasm, and mitochondria. -tubulin and TIM23
were used as markers for subcellular localization in the cytoplasm and mitochondria,
respectively.
Supplementary Figure S6
Reduced expression of BMCC1 in epithelial-derived
prostate and colon cancers. Expression levels of BMCC1 in prostate (a) and colon
cancer (b) tissues (T) and their corresponding normal tissues (N) were demonstrated by
IHC (prostate cancer; T=5 and N=4 cases, colon cancer; T=5 and N=5 cases). Origin
and clinical information, such as stage, Gleason score and TNM, were provided. (a)
Expression levels of BMCC1 in normal prostate tissues (N-1 to N-3) corresponded to
those of the tumors (T-1 to T-3). (b) Two mucinous adenocarcinoma (T-1 and T-2) and
three adenocarcinoma (T-3, T-4 and T-5) tissue sections derived from ascending colon,
rectum and sigmoid colon were analysed in the patients with T3N2 or T4N1 tumors
with lymph node metastasis. Patients with T-5 possessed distant metastases including
lymph node. The corresponding normal colon tissues of three tumor cases, T-1 to T-3,
are listed as N-1 to N-3.