Weise and Dünker
TFF1 in retinoblastoma cells
Supplementary material and methods
Semi-quantitative PCR analysis and Real-time PCR
For semi-quantitative PCR analysis of TFF2 and TFF3 expression in Rb cells or human cDNA the following primer
pairs were used. TFF2: 5’-TGCAGCTGAGCTAGACATGG -3’ and 5’-AACACCCGGTGAGCCAGATG -3’; TFF3: 5’GTGGTCATGGCTGCCAGAG -3’ and 5’-AGCTGGAGGTGCCTCAGAAG -3’. Primer pairs were used in 50 µl reactions
and the following program was applied: 2 minutes at 94°C, 45 cycles of 30 seconds at 94°C, 15 seconds at 55°C,
30 seconds at 72°C, 5 minutes at 72°C. The specificity of all three TFF primer pairs was proven in a control PCR
using cDNA derived from human stomach tissue (kindly provided by E. Cario) as positive control.
Quantitative Real-time PCR analyses were performed using the following Taqman Gene Expression
Assay (Applied Biosystems) for cyclin D1: Hs00277039_m1. GAPDH (Hs99999905_m1) was used as an
endogenous control. Real-time PCR reactions were performed in duplicate and a total volume of 20 µl was
applied to the following program: 2 minutes at 50°C, 10 minutes at 95°C, 40 cycles of 15 seconds at 95°C and 60
seconds at 60°C.
Western blotting
WERI-Rb1 and Y-79 cells were serum-starved or grown in medium containing 10% FCS for 72 h. Total protein
extract was separated on a SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and
membranes were blocked in Tris buffer saline (200 mM Tris, pH 7.6; 1.5 M NaCL) with 1% Tween-20 (TBS-T)
containing 5% skim milk powder for 60 min. For detection of cyclin D1 a mouse monoclonal anti-cyclin
D1antibody (1:2,000; DCS6; Cell Signaling) was applied at 4°C overnight diluted in TBS-T buffer supplemented
with 0.025% BSA and 0.02% sodium acid. Visualisation of antibody-antigen-complexes was achieved with a
horse radish peroxidase-coupled antibody diluted 1:10,000 in TBS-T buffer containing 2% skim milk powder at
room temperature for 30 min. Actin, detected by a goat polyclonal anti-actin antiserum (1:1,000; sc-1616;
Santa Cruz), served as an internal standard to verify equal protein loading in all lanes.
CDK/cyclin co-immunoprecipitation experiments in HEK293 cells
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For transient transfections in HEK293 cells CDK6 and CDK2 cDNAs were cloned into a pIRES-hrGFP-2a vector
(Clontech) using EcoRI and XhoI as restriction enzymes to fuse the CDKs into the reading frame of the human
influenza hemagglutinin (HA) tag for later immunoprecipitation by anti-HA antibodies. Human cDNAs for cyclin
D1, cyclin E1 and TFF1 were cloned into the pCS2+ expression vector using EcoRI and XhoI as restriction
enzymes. The following primers were used to amplify the cDNAs with pfu Ultra II polymerase (Stratagene) from
human stomach cDNA for cloning into the expression vectors mentioned above (restriction sites underlined):
CDK6: 5’-ATATGAATTCATGGAGAAGGACGGCCTGTGC-3’, 5’-ATATCTCGAGGGCTGTATT CAGCTCCGAGGTG-3’;
CDK2:
5’-ATATGAATTCATGGAGAACTTCCAAAAGGTG-3’,
5’-ATATCTCGAGGAGTCGAAGATGGGGTACTGG-3’;
cyclinE1: 5’-ATATGAATT CATGCCGAGGGAGCGCAGGGAG-3’, 5’-ATATCTCGAGTCACGCCATTTCCGGCC CGCTGCTC3’; cyclinD1: 5’-ATATGAATTCATGGAACACCAGCTCCTGTGC-3’, 5’-ATATCTCGAGTCAGATGTCCACGTCCCGCAC-3’;
TFF1: 5’-GGTCGAATTCGGAG CAGAGAGGAGGCAATG-3’, 5’-AGTCCTCGAGGGCAGATCCCTGCAGAAGTG-3’. All
expression constructs were sequence verified. For co-immunoprecipitation experiments, 3 x 106 HEK293 cells
were transfected with suitable combinations of expression vectors for CDK6-HA (5 µg), CDK2-HA (5 µg), cyclin
D1 (5 µg), cyclin E1 (5 µg), TFF1 (2.5, 5.0, and 7.5 µg) and empty expression vector (as required to keep amount
of transfected DNA constant) by the calcium-phosphate co-precipitation method. After 48 h, whole cell extracts
were made from transfected HEK293 cells by lysing cells in RIPA buffer (supplemented with additives) for 30
min on ice. After clearing the lysates by centrifugation at 16,000 x g at 4°C for 10 min, the protein
concentration of the extracts was determined using a BCA protein assay kit (Thermo Scientific). For
immunoprecipitation 500 or 1,000 µg of total protein were combined with 0.5 µg anti-HA antibody (3F10,
Roche applied Science) and 25 µl of a 50% protein-G-sepharose beads (Roche) suspension in RIPA buffer and
incubated on a rotary shaker at 4°C for 3 h. After extensive washing immunoprecipitates were eluted from the
beads with SDS-PAGE sample buffer and analysed by SDS-PAGE and Western blotting. Following SDS-PAGE,
proteins were transferred onto nitrocellulose membrane and membranes were blocked in TBS-T buffer
containing 5% skim milk powder for 60 min. For detection of proteins the following primary antibodies were
used with the dilutions indicated: rat monoclonal anti-HA antibody (1:1,000; 3F10; Roche Applied Science);
rabbit monoclonal anti-estrogen inducible protein pS2 antibody (1:2,000; ab92377; abcam); mouse monoclonal
anti-cyclin D1 antibody (1:2,000; DCS6; Cell Signaling); mouse monoclonal anti-cyclin E1 antibody (1:1,000;
HE12; abcam); goat polyclonal anti-actin antiserum (1:1,000; sc-1616; Santa Cruz). If not indicated otherwise,
primary antibodies were applied at 4°C overnight in TBS-T buffer supplemented with 0.025% BSA and 0.02%
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sodium acid. Visualisation of antibody-antigen-complexes was achieved with appropriate species-specific horse
radish peroxidase-coupled antibodies diluted 1:10,000 in TBS-T buffer containing 2% skim milk powder at room
temperature for 30 min.
Fig. S1 Expression of TFF1, TFF2 and TFF3 in healthy human retina and different retinoblastoma cell lines as
revealed by semi-quantitative RT-PCR. Specificity of the TFF primers was tested using cDNA from human
stomach tissue as a positive control (a). The 100 bp molecular ladder standard is shown on the left. The 200 bp,
300 bp and 600 bp lanes are indicated for orientation. The three TFF peptides exhibit strikingly different and
partially opposed (TFF1 vs TFF3) expression profiles (b). In the healthy human retina only TFF3 mRNA was well
detectable, whereas in retinoblastoma cell lines only weak TFF3 expression was observed. TFF2 transcripts
were neither detectable in human retina nor in retinoblastoma cell lines. TFF1 expression was detectable in
different retinoblastoma cell lines at variable levels, but not in the healthy human retina. The integrity of the
cDNAs was tested by amplification of the GAPDH transcript. -RT: cDNA synthesis reaction without reverse
transcriptase.
Fig. S2 Cyclin D1 expression levels in WERI-Rb1 and Y-79 cells under growth arrest conditions by serum
starvation as revealed by Real-time PCR (a) and Western Blot analyses (b). Comparing cells serum-starved (FCS) or grown in medium containing 10% serum (+ FCS) for 72 h, growth arrest was clearly demonstrated by a
significant down regulation of cyclin D1 in the absence of FCS (a). Cyclin D1 was not detectable in immunoblots
of WERI-Rb1 cells, but in Y-79 cells serum starvation (- FCS) resulted in a clear decrease of the cyclin D1 content
(b). Actin served as an internal standard to verify equal protein loading in all lanes. Numbers on the left indicate
positions and sizes of protein markers.
Fig. S3 TFF1 inhibits CDK6-cyclin D1 interactions as revealed by co-immunoprecipitation. HEK293 cells were cotransfected with equal amounts of expression constructs for CDK6-HA and cyclin D1 (a) or CDK2-HA and cyclin
E1 (b) and increasing amounts of TFF1 expression plasmid DNA. TFF1 inhibits CDK6-cyclin D1 coimmunoprecipitation in transiently transfected HEK293 cells (a), whereas co-precipitation of cyclin E1 by CDK2HA protein was not impaired by TFF1 expression (b).
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