Figure S1
A. ASAP1 expression in SW1116 (SW) and LS174T (LS) human colorectal tumor cell
lines. RT-PCR was performed using cDNA derived from the cells as template. To this
end, total RNA was isolated using TRIZOL (Invitrogen) and used to generate cDNA
using Superscript II (Invitrogen). RT-PCR was performed using the following primers:
huASAP1F: 5’-GCG GAT CCG CTG GCC AAG AAT GTA GGA AAC AAT AG-3’
and huASAP1R: 5’-GCG AAT TCG GGA AAG CAG ATC TTC ACA CTG GG-3’
(amplifies nucleotides 977-1532 of human ASAP1 (KIAA1249)). Samples were
amplified with primers for ASAP1 or HPRT for a control. Hundred base pair markers
(M) are shown for reference. The most intense marker band is 600bp.
B. Western blot analysis of ASAP1 expression in SW1116 (SW) and LS174T (LS) cell
lines. Cell lysates were western blotted and probed with 7B12 anti-ASAP1 antibodies.
C. Immunohistochemical staining for ASAP1 of a section of a SW1116 tumor grown in
nude mice. The section was stained with 7B12 (red colour) and counterstained with
hematoxylin (blue colour).
Figure S2
A. 1AS clones used in tumor experiments in vivo. 1AS cells were stably transfected with
empty vector (vector), flag-tagged dominant negative ASAP1 (dom neg) or with flagtagged ASAP1 (ASAP1). Clones were checked for expression of ASAP1 using western
blotting and probing with anti-flag antibodies. The clones shown in this western blot
probed with the flag tag antibody had the highest ASAP1 expression and were selected
for the tumor experiments.
B. ASML clones used in tumor experiments in vivo. ASML cells were stably transfected
with empty vector (vector) or with flag-tagged dominant negative ASAP1 (dom neg).
Clones were checked for expression of ASAP1 using western blotting and probing with
anti-flag antibodies. The clones shown in this western blot probed with the flag tag
antibody had the highest ASAP1 expression and were selected for the tumor experiments.
C. ASAP1 transient transfections. In all experiments in which ASAP1 (wild type or
dominant negative) was transfected into cells, expression was assured using western blot.
This panel shows an example of such a blot. 1AS cells were transiently transfected with
empty vector (V), flag-tagged dominant negative ASAP1 (D) or with flag-tagged ASAP1
(A). Lysates were checked for expression of ASAP1 using western blotting with anti-flag
antibodies.
Figure S3
Expression of ASAP1a/b and ASAP11c in a panel of rat tissues assessed by semi-quantitive PCR.
A. Design of the PCR strategy. The position of the primers used to amplify ASAP1 from the
various tissues are indicated relative to the domain structure of ASAP1 (see Figure 3 for details of
domain structure). The position of the primers allows the ASAP1a and ASAP1b isoforms to be
distinguished from the ASAP1c isoform on the basis of the size of the PCR product. Specifically,
PCR amplification of ASAP1a and ASAP1b results in products with identical size (249 nt), while
the ASAP1c amplicon is smaller (213 nt). B. ASAP1 RT-PCR performed on cDNA derived from
the indicated tissues or 1AS or ASML cells using the diagnostic primers. Amplification of hprt
served as a loading control. For a negative control, water instead of cDNA was added to the PCR
reaction (H2O). Total RNA was isolated using TRIZOL (Invitrogen) or PeqGold (Peqlab,
Erlangen) and used to generate cDNA using Superscript II (Invitrogen). Rat ASAP1 isoforms
were amplified using the following primers: rASAP1onco2F: 5’-TCC TCT CTG CAG CTT GAT
CCG A-3’
and rASAP1onco2R: 5’-AGG CTG CCT GTT GGA AGT TGC-3’
(amplifies nucleotides 883-1132 of rat ASAP1a (DQ238622) and rat ASAP1b (DQ238623), and
nucleotides 883-1095 of rat ASAP1c (DQ238624)). PCR was performed under standard
conditions using 30 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec.
Hprt served as a loading control using the following primers:
rHPRT512 fw: 5’-TGG TCA AGC AGT ACA GCC CC-3’
rHPRT772 rev: 5’-TAC TGG CCA CAT CAA CAG GA-3’
(amplifies nucleotides 512-772 of rat hprt1 (NM_012583)).
Table 1. Genes from the MLSSH and PLSSH libraries that are upregulated more
than 2-fold in MatLyLu cells compared to G cells. Custom microarray chips of all 268
genes were differentially hybridized with fluorescently labeled probes from MatLyLu and
G cells. Fold upregulation in the MatLyLu cells compared to G cells was calculated. The
mean of a least three independent data points is shown. Of the 268 genes tested,
expression of only 16 could not be evaluated due to poor spot quality.
Gene Name
Accession no.
HMG-1
Thrombospondin
CINC-2 alpha
CD24
Novel clone # 143
Novel clone # 32
Caveolin 1
Calcyclin
Novel clone # 175 (Klon 185)
Novel clone # 176 (Klon 225)
Novel clone # 132
Hai2/Spint2
Ribosomal protein S13
Novel clone # 101
Ribosomal protein S7
GPR2
h-Cadherin
Oncomodulin
NADP-dependent isocitrate dehydrogenase
Caveolin 2
ALEX3
Supervillin
Ribosomal protein L26
UPA
Lipocortin I
Calpactin I
U6 snRNA associated Sm-like protein
Novel clone # 35
Capping protein alpha 2
Novel clone # 80
Cytokeratin-8
Novel clone # 142
Ovarian cathepsin B
Novel clone # 71
Calmodulin
X62875
S79301
D87926
U49062
AF125348
X52278
AF099016
X53378
X53377
U13667
U59289
J02705
L35317
AF035752
BC005194
XM_030478
X14671
X65651
Y00446
X66871
AF182294
U16741
S76054
AF057143
X13933
Fold upregulated in
MatLyLu compared
to G cells
14.7
14.2
9.8
7.8
7.4
7.3
6.9
6.7
6.4
6.3
5.7
5.6
5.3
5.0
4.3
3.8
3.8
3.7
3.7
3.7
3.7
3.4
3.4
3.1
3.1
3.1
3.0
2.9
2.9
2.8
2.8
2.8
2.7
2.7
2.6
Novel clone # 113
Ribosomal protein S24
Pyruvate kinase
Metastasis-associated factor
Claudin-6
Calgizzarin
Novel clone # 131
Ezrin
Novel clone # 29
ASAP1
Mitochondrial oxidase c
KS1/4 antigen (EGP-protein homolog)
Novel clone # 42
SUP
Prothymosin alpha
Novel clone # 39
AHNAK
Novel clone # 14
Tropoelastin gene
SSAT
Poly-A binding protein
Novel clone # 170 (Klon 96)
Novel clone # 169 (Klon 86)
CDC10
Novel clone # 26
Novel clone # 58
TGF alpha
RAD52 gamma
IQGAP2
Novel clone # 95
Novel clone # 158
Novel clone # 130
Megakaryocyte potentiating factor
X51538
J04459
AJ001043
NM_018777
D38583
X60671
AF075461
M27315
M76124
AB033771
X56135
M80899
M85178
M55580
X65553
AF142759
M31076
AF125949
U51903
D86370
2.6
2.5
2.5
2.5
2.5
2.5
2.5
2.4
2.4
2.3
2.3
2.3
2.3
2.3
2.3
2.2
2.2
2.2
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.0
2.0
2.0
2.0
2.0
2.0
2.0
Methods:
Production of custom microarray chips. The clones of the MLSSH (Nestl et al., 2001) and
PLSSH (von Stein et al., 1997) libraries were amplified by PCR and spotted on glass slides
covered with the substrate CMT-GAPS (Corning Lifesciences). Each clone was spotted eight
times on each array. As controls HPRT, GAPDH and -actin PCR products were used.
Screening of microarray chips. Polyadenylated RNA was prepared using the Purist mRNA kit
(Ambion) and fluorescently labeled with Cy3 or Cy5 using the CyScribe First Strand cDNA kit
(Amersham). Probes were combined, denaturated at 95°C for one minute, then mixed with DIGEasy Hyb (Roche) hybridisation buffer. The probes were incubated with the microarrays at 42°C
for 12h. Hybridised arrays were washed at 42°C in 2 x SSC; 0.1 % SDS (10 min), 0.1 x SSC; 0.1
% SDS (5 min), 0.1 x SSC (4 min) and 0.01 x SSC (15 sec). The arrays were scanned with the
Axon GenePix 4000B (Axon Instruments) using two different signal amplifications (high, low).
The results were analysed using GenePix Pro (Axon Instruments).
Table 2. Immunohistochemical staining of human tumors and the corresponding nonneoplastic tissue with anti-ASAP1 antibodies.
Organ
Normal tissue
Tumor tissue
Negative except for strong staining
of the parietal cells
Epithelium +/-
Moderately differentiated adeno2/9 +/++
carcinoma
Squamous cell carcinoma
4/9 ++/+++
Alveoli -, bronchi +/Some sections showed staining of
the luminal epithelium, crypts -
Squamous cell carcinoma
Thyroid
Negative
Papillary carcinoma
Kidney
Proximal convoluted tubules +
Renal cell carcinoma
Endometrium
Epithelium +++
Adeno-carcinoma
Gall bladder
Epithelium +
Adeno-carcinoma
Larynx
Negative
Squamous cell carcinoma
Cervix
Epithelium +++, especially basally
Squamous cell carcinoma
Lymph nodes
Heterogeneous +
Lymphoma
Skin
Epidermis ++
Malignant melanoma
Breast
Epithelium +
Infiltrating ductal carcinoma
Liver
+/-
Hepatocellular carcinoma
Bladder
Epithelium +
Transitional cell carcinoma
Ovary
Negative
Serous cystadenocarcinoma
Pancreas
+/Ductal adeno-carcinoma
Epithelium heterogeneous, from - to
Adeno-carcinoma
++
Stomach
Esophagus
Lung
Colon
Prostate
Adeno-carcinoma
- no staining; + weak staining; ++ moderate staining; +++ strong staining.
Positive
0/7
4/10 ++
3/10 +
4/10 ++/+++
4/10 +
2/8 ++/+++
5/10 +++
2/10 ++
2/10 +
4/10 ++/+++
5/9 ++
1/9 +
6/10 +
5/10 ++
2/10 +
3/10 ++
2/10 +
5/10 ++
3/10 +
2/10 +
3/8 +/++
4/10 ++
4/10 +
0/10
4/9 ++
1/9 +