Supplementary Materials and Methods
Materials
Horseradish peroxidase-conjugated antibodies for mouse (sc2005) and rabbit IgG
(sc2004) were from Santa Cruz Biotechnology (USA). TRITC-conjugated phalloidin
and mouse monoclonal antibody specific for -actin (A5441) were from Sigma
Chemical (USA). Alexa fluor 488/555-conjugated IgG antibodies were from
purchased from Alexis Corp. (Carlsbad, CA). Wortmannin, and antibodies specific
for phospho-Akt (#9271), PTEN (#9559) and Akt1 (#9272) were from Cell Signaling.
PTEN siRNA was from Ambion. An ATP assay kit was from Abcam. N-C6-Derythro-ceramide
and
C12-glucosylceramide
were
from
Matreya
(USA).
Hoechst33342 was from Dojindo (Japan). Recombinant human EGF was from
Peprotech (USA). Halttm Phosphatase Inhibitor Cocktail was from PIERCE (USA).
Human p110 cDNA was purchased from Kazusa DNA research institution.
Cell culture
SKOV3 cells were kindly provided from Dr. Carla Grandori (Fred Hutchinson Cancer
Research Center, Seattle, WA) and grown in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% FBS. Those cells were authenticated by JCRB
Cell Bank (Osaka, Japan). OSE4 cells cultured in DMEM supplemented with 10%
FBS were gifted from the Department of Obstetrics and Gynecology, Kumamoto
University. Cells were maintained at less than 80% confluence under standard
incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37 ˚C). No
mycoplasma contamination was observed in all cell lines.
Cell migration assay
Transwell cell migration assay was performed according to modified methods
described by Asano et al.1 SKOV3 cells (1 × 106), grown on 10-cm dishes, were
treated with C6-ceramide for 3 h, and then harvested. An appropriate volume of
DMEM supplemented with 10% FBS in the absence or presence of C6-ceramide was
introduced into the lower wells, and cells (1 × 105) were plated in the top well. The
Transwell chambers were incubated at 37 °C and 5% CO2 for 6 h. Cells on the upper
surface of the membrane were scraped off and migratory cells attached to the lower
surface were stained with Hoechst33342 and counted.
Immunofluorescence
Cells, growing on glass bottom dishes, were fixed for 10 min at room temperature
with 4% formaldehyde in phosphate-buffed saline (PBS) and washed with PBS. Next,
cells were treated for 10 min with 0.1% TritonX-100, washed with PBS, and blocked
for 1 h with PBS containing 2% human serum. The primary antibodies for Akt or V5
were diluted in PBS containing 2% human serum, and incubated for overnight at 4°C.
Samples were washed with PBS, and Hoechst33342 and/or TRITC- or FITCconjugated anti-IgG antibodies were applied for 1 h in PBS containing 2% human
serum. Confocal laser microscopy was performed using an LSM780 confocal
microscope (Carl Zeiss, NY).
Immunoblotting
Cells were washed three times with PBS supplemented with Halttm Phosphatase
Inhibitor Cocktail and then lysed using Laemmli buffer. The protein samples (20 µg)
were subjected to SDS-polyacryamide gel electrophoresis (4–20% gradient gels).
Proteins were electrophoretically transferred to nitrocellulose membranes, blocked
with PBS/0.1% Tween 20 (PBS-T) containing 5% nonfat dried milk, washed with
PBS-T, and incubated with antibodies for Akt1 (1 to 1,000), phospho-Akt (1 to
1,000), and PTEN (1 to 500) in PBS-T containing 5% nonfat dried milk. The blots
were washed with PBS-T and incubated with secondary antibody conjugated with
horseradish peroxidase in PBS-T containing 5% nonfat dried milk. Detection was
performed using enhanced chemiluminescence reagent, and the quantification of the
chemiluminescent signals was performed with a digital imaging system (VersaDoc,
Bio-Rad).
Transfection with small interference RNAs (siRNAs)
Cells (2  105 cells/60 mm dish) were transfected with 10 nM siRNAs for PTEN
using RNAiMax Transfection Reagent (Invitrogen) according to the manufacture’s
instructions. After 48 h, transfection reagents were washed out and cells were
incubated with 10% FBS.
Preparation of the p110 delta expression construct
Human p110 cDNA wwas amplified by PCR using the sets of primer, BamHIstart1F (ctgcagggatccatgccccctggggtg) containing BamHI site and XhoI-nostop3132R
(ggtaccctcgagctgcctgttgtctttggacac) containing XhoI site without stop codon. PCR
products and pcDNA3.1 / V5-His A (Invitrogen) were digested with BamH I/ XhoI
and each fragment were ligated. Sequence of produced plasmid was verified with T7
primer and BGH primer as universal primer.
Determination of ceramide interacting proteins
SKOV3 cells transfected with V5-tagged p110 pcDNA3.1 plasmid vectors were
lysed in RIPA buffer (100 mM NaCl, 1% Triton-X 100, 1% sodium deoxycholate,
0.1% SDS, 1 mM EDTA, 10 mM Tris HCl, pH 7.5) at room temperature. The lysates
were centrifuged at 20,000  g for 10 min, and then the supernatants (1 mg/ml
protein) were treated with 10 µM biotin or C6-ceramide-conjugated biotin in the
presence of streptavidin-conjugated magnetic beads at room temperature for 1 h. The
magnetic bead complexes were washed three times with RIPA buffer, and C6ceramide-interacting proteins were extracted. The proteins were subjected to
immunoblotting using antibodies against V5.
Supplementary Table 1. Effects of sphingolipids on in vitro kinase activity.
IC50 values of C6-ceramide and C16-glucosylceramide for individual kinases were
determined. C6-ceramide had a stimulatory effect on Akt2, Akt3, and PDK2, and the
percentage increases at 100 µM C6-ceramide were shown. N.D., not determined.
Experiments were performed by ProQinase GmbH (Freiburg, Germany).
Protein or lipid
kinase
Akt1
Akt2
Akt3
EGF-R wt
mTOR
PDK1
PI4K2A
PI4K2B
PI4KB
PIK3C2A
PIK3C2B
PIK3C2G
PIK3C3
PI3KCA
PI3KCB
PIK3CG
PIK3CD/PIK3R1
PIP5K1A
PIP5K1C
C6-Ceramide
(µM)
> 100
Over 200% increases at 100
µM
Over 300% increases at 100
µM
> 100
> 100
Over 150% increases at 100
µM
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
C16-Glucosylceramide
(µM)
38
> 100
76
2.3
N.D.
16
> 100
> 100
N.D.
> 100
> 100
N.D.
N.D.
> 100
> 100
N.D.
N.D.
N.D.
N.D.
Reference
1.
Asano S, Kitatani K, Taniguchi M, Hashimoto M, Zama K, Mitsutake S,
Igarashi Y, Takeya H, Kigawa J, Hayashi A, et al. Regulation of cell migration by
sphingomyelin synthases: sphingomyelin in lipid rafts decreases responsiveness to
signaling by the CXCL12/CXCR4 pathway. Mol Cell Biol 2012; 32:3242-52.