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SUPPLEMENTAL MATERIALS AND METHODS
Generation of transgenic fly strains
The PAT1 cDNA in pSPORT-SfiI (DKFZ p686A1551Q [RZPD]; Wellenreuther et
al., 2004) and the PAT4 cDNA in pBluescriptR (IMAGE 5313230) were subcloned in
to the transformation vector pUAST (Brand and Perrimon, 1993) following digestion
at restriction sites flanking the open reading frames of the cDNA clones namely,
EcoRI and BglII (PAT1) and NotI and SpeI (PAT4).
UAS-PAT1 and UAS-PAT4 transgenic flies were generated in the lab by
microinjection of fly embryos expressing 2,3 transposase (y w; Ki pp 2,3 flies were
kindly provided by Hugo Bellen).
Fly genetics and phenotypic analysis
Fly strains containing the following GS transposable element insertions (Toba et al.,
1999) were used: pathGS13857 to overexpress path and foxoGS9928 to overexpress the
pro-apoptotic gene, foxo (gifts from Toshiro Aigaki). Other fly stocks, genetic crosses
and the method for ommatidial size measurement were as described in Goberdhan et
al., 2005. Flies were cultured on standard cornmeal agar food at 25ºC.
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Cell culture conditions
The following human cell lines were obtained from the American Type Culture
Collection (ATCC): pancreatic carcinoma (AsPC-1), cervical cancer (HeLa), prostate
carcinoma (DU-145, PC3, LNCaP), lung carcinoma (NCI-H460, A549), breast cancer
(MDA468), hepatocellular liver carcinoma (HepG2) and epithelial colorectal
adenocarcinoma (Caco-2) and human embryonic kidney (HEK-293) cells. Cancer
Research UK Cell Services supplied the following human cell lines: embryonic lung
fibroblasts (W138) and breast adenocarcimoma (MCF-7). The MCF-7 cells were
cultured in RPMI (GibcoTM Invitrogen) medium supplemented with 10% FCS
(GibcoTM Invitrogen) and the HEK-293 cells were cultured in 10% FBS, 1 x NEAA, 4
mM L-Glutamine, and incubated at 37ºC, 5% C02. 1% Penicillin/Streptomycin
(Invitrogen) was added as an antibiotic to all media.
Each siRNA test was repeated in triplicate from independent wells. Validated siRNA
primer duplexes (Eurogentec) were employed targeting human mTOR (si825 5′AGGACGCUCACAUUGCUAGAUGUGG-3′,
5′-
si826
ACCUCAAGCAAAGUAGGGUACUUCC-3′
and
si827
5′-
UUAAGGAUCAACAAGGCUCCAUGGA-3′),
PAT1
(si158
5′-
CCUGCAAUUUGGAGCUAAU-3′,
and
si160
si159
5′-GCGCUUUGGUCAAAGCAAU-3′
5′-AUGCUCCCAUCUUCAUCAA-3′)
and
PAT4
(si435
5′-
UAAUGAGGCCCUUGAUAAA-3′, si436 5′-UCAGAAGCAUUACCAACUU-3′,
si437 5′-GGCCCUUGAUAAAUGAGCA-3′), with a scrambled siRNA sequence
(Eurogentec), lipofectamine or magnet-assisted transfection alone and non-transfected
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cells as negative controls. The final concentration of siRNA used in each well was
100 nM for MCF7 cells and 20 nM for HEK-293 cells.
Q-RT-PCR
Total RNA extraction from cells was performed using RNeasy® kits (Qiagen)
following the manufacturer’s instructions, and quantified with a Nanodrop® 1000
(Thermoscientific). The High Capacity cDNA reverse transcription kit (Applied
Biosystems) was employed for reverse transcription of RNA into cDNA. Tissue
cDNAs were supplied by PrimerDesign.
Q-RT-PCR was carried out using the Applied Biosystems 7000 PCR system,
Precision SYBR® Green mastermix (PrimerDesign) and Quantitect® Q-RT-PCR
primers (Qiagen) that were specific for all the target and housekeeping genes
analysed. The comparative cycle threshold method (ΔCT) was employed for data
analysis. For a specific cell line or tissue, the Ct values for both PAT1 and PAT4 were
normalized against the Ct value of the HPRT1 housekeeping gene, i.e. Ct [HPRT1] –
Ct [PAT1 or PAT4] and the percentage difference relative to HPRT1 was then
calculated. For HEK-293 cells, the ubiquitin C gene (PrimerDesign) was used as the
housekeeping gene control, since it more accurately reflected the levels of total
cDNA.
Immunostaining
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Fixed HEK-293 cells fixed with 4% paraformaldehyde were rinsed three times with
PBS and then placed in blocking buffer (0.25% BSA, 0.05% Triton-X in PBS) for 1 h
at room temperature. Anti-PAT1 antiserum (1:1000; Wreden et al., 2003) was diluted
in blocking buffer before being applied to cells. The plates were incubated for 1h at
room temperature. The primary antibody was removed and the cells were washed for
3 x 5 min in blocking buffer. Secondary antibodies were diluted in blocking buffer,
which also included DAPI (0.1 µg/ml) and in some cases a 1:1000 dilution of a
TRITC-phalloidin conjugate (1 g/ml; Sigma). The cells were incubated for 1 h at
room temperature, washed for 3 x 5 min before mounting in CitifluorTM anti-fading
mountant. Slides were sealed with varnish and stored in the dark at 4ºC until imaged
by confocal microscopy.
Western blots
Protein was quantified using a Bradford assay (Bio-Rad). The readout was performed
in PerkinElmer plate reader at 586 nm. All protein extracts were stored at -20ºC. 40
g of each protein lysate were routinely loaded on a 10% polyacrylamide gel. For
immunoblotting, mouse anti-Phospho-Thr389-p70 S6K1 (Cell Signalling Technology
[CST]; 1:1000 in 2% Marvel), rabbit anti-Phospho-Ser473-Akt (CST; 1:2000 in 5%
BSA), rabbit anti-Phospho-Thr24-Fox01/Thr32-Fox03a (CST; 1:1000 in 5% BSA),
rabbit anti-mTor (CST; 1:1000 in 5% BSA), rabbit anti-Phospho-Ser240/244-PS6,
rabbit anti-Phospho-Ser65-4E-BP1, rabbit anti-S6, rabbit anti 4E-BP1 (all CST all
1:1000 in 5% BSA), rabbit anti PAT1 (Wreden et al., 2003; 1:10000 in 5% Marvel)
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and mouse anti--tubulin (Sigma; 1:5000 or 1:10000 in 1% or 5% Marvel
respectively) were used. All dilutions were prepared in TBS (Tris buffered saline)
0.1% Tween® 20 (Sigma-Aldrich). After incubation with appropriate secondary
antibodies signals were visualised using the ECL™ western blot detection kit
(Amersham).
References
Brand, AH, Perrimon, N. (1993). Targeted gene expressionas a means of altering cell
fates and generating dominant phenotypes. Development 118: 401-15.
Wellenreuther, R, Schupp, I. The German DNA Consortium, Poustka, A, Wiemann,
S. (2004). SMART amplification combined with cDNA size fractionation in order to
obtain large full-length clones. BMC Genomics 5: 36.
Wreden, CC, Johnson, J, Tran, C, Seal, RP, Copenhagen, DR, Reimer, RJ, Edwards,
RH. (2003). The H+-coupled electrogenic lysosomal amino acid transporter LYAAT1
localizes to the axon and plasma membrane of hippocampal lneurons. J Neurosci 23:
1265-1275.
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SUPPLEMENTARY FIGURES
Supplementary Figure 1. Transcript levels of PAT1, PAT4 and mTOR are strongly
reduced following siRNA knockdown in MCF-7 breast cancer cells. Transcript levels
were measured by Q-RT-PCR in triplicate 120 h after transfection from cells treated
with PAT1 siRNAs (si158, si159 and si160), PAT4 siRNAs (si435, si436 and si437)
and mTOR siRNAs (si825, si826 and si827), compared to transcript levels from
control cells treated with a scrambled siRNA control (sc), lipofectamine only (lp) and
non-transfected (nt). All knockdowns produced a significant reduction in the level of
the targeted transcript (P<0.02; error bars represent SEM). Significant reductions in
PAT1 and PAT4 transcripts were observed within 24 h of siRNA transfection (left
panel).
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Supplementary Figure 2. PAT1, PAT4 and mTOR siRNA knockdown reduce protein
and transcript levels in HEK-293 cells. (A-C) Transcript levels measured by Q-RTPCR in triplicate samples 72 h after transfection. Cells were treated with mTOR
(si825, si826 and si827; A), PAT1 (si158 and si159; B) and PAT4 (si435 and si437;
C) siRNAs and compared to transcript levels from control cells treated with a
scrambled siRNA (sc) and with only MATra. All knockdowns produced a significant
reduction in the level of the targeted transcript (*P<0.01; **P<0.001; error bars
represent SEM). (D, E) Blots of protein lysates from cells 72 h after transfection.
Lysates from cells treated with mTOR (si825 and si826) siRNAs were compared to
control lysates treated with sc siRNA or MATra alone, by probing with an anti-mTOR
antibody (D). Lysates from cells treated with PAT1 (si158 and si159) siRNAs were
compared to control lysates treated with sc siRNA or MATra alone, by probing with
an anti-PAT1 antibody (Wreden et al., 2003; E).
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