Supplementary Information
Supplementary Figure Legends
Supplementary Figure 1. RasGRP3 expression in tumor cell lines and melanoma tumors. (A) Detection of RasGRP3 expression in human melanoma tissues by in situ hybridization. Tissue microarrays (Biomax Inc) were hybridized with antisense probes 1 and 2 generated against two non-overlapping regions of the RasGRP3 mRNA. The bright and dark field pictures (left and right panels, respectively) show labeled cells within the tissue samples #B4 and #D5 of the array. Silver grains, black dots in bright field and white dots in dark field, deposited over the cells indicate that radiolabeled molecules are hybridized to endogenous mRNA. Two sense probes were used as control. (B) Q-PCR analysis of RasGRP3 gene expression in a melanoma cDNA array (TissueScan Melanoma Tissue pPCR Panel I, Origene). Beta actin gene expression was used as an internal control. Values are normalized with the mean of normal skin and represent average + SEM of three independent experiments. (C) RT-PCR analysis of RasGRP3 gene expression in multiple cell lines. RT-PCR results were confirmed by DNA sequencing. Ramos cells were included as a positive control. GAPDH gene expression was used as an internal control. Results are representative of 3 independent experiments.
Supplementary Figure 2. Effects of inhibiting RasGRP3 expression by shRNAs (A) RasGRP3-LNCaP cells were transduced with lentivirus containing RasGRP3 shRNAs 123, 236, 740, 772, 854, 1255, 1258, 1426 and two scrambled control shRNAs. The RasGRP-V5 expression level was detected after 120 hours by immunoblotting. The b-actin levels were determined as a control for equal loading. (B) M14 cells were transduced with lentivirus containing RasGRP3 shRNAs 123, 236, 854, 1426 and two scrambled control shRNAs. The RasGRP3 expression level was detected after 96 or 144 hours by RT-PCR. The GAPDH levels were determined as a control for equal loading. All results in this figure were representative of 2 independent experiments.
Supplementary Figure 3. Inhibition of endogenous RasGRP3 expression inhibited anchorage independent growth. (A and B) Colony formation of M14 and SK-MEL-5 cells in soft agar. Digital pictures were taken 10 days after plating the cells in soft agar. All results in this figure were representative of 3 independent experiments.
Supplementary Figure 4. AKT phosphorylation and its effects on cell proliferation. (A) Primary melanocytes and multiple melanoma cell lines cultured with 10% FBS were treated with/without HGF (20 ng/ml), EGF (100 pg/ml), VEGF (5 ng/ml), TGF-b (5 ng/ml), or TNF-α (100 pg/ml) for 20 minutes. Phospho-AKT was detected by immunoblotting of cell lysates. (B) The cells were treated with 20 µM AKT inhibitor V (Triciribine) for 48 hours and analyzed for cell proliferation using the CyQuant NF cell proliferation assay and for phosphorylation of AKT by immunoblotting. Values in panel B represent the mean ± SEM of three independent experiments. All other results in this figure were representative of 3 independent experiments.
Supplementary Figure 5. Endogenous RasGRP3 contributes to Ras and Akt1/2/3 activation in multiple melanoma cell lines. (A) Individual cell lines were transiently transfected with RasGRP3 siRNA pool and Control siRNA pool 1 at 80 nM final concentration. After 72 hours, cells were treated with or without HGF (20ng/mL) and EGF (100 pg/ml) as indicated for 20 minutes. RasGRP3, Akt and phosphorylated Akt were detected by immunoblotting of cell lysates. (B) SK-MEL-2 and SK-MEL-28 cell lines were transiently transfected with RasGRP3 siRNA pool and Control siRNA pool 1 at 80 nM final concentration. After 72 hours, cells were treated with or without HGF (20ng/mL) as indicated for 20 minutes. RasGRP3, Akt and phosphorylated Akt were detected by immunoblotting of cell lysates. (C) The Tet-on stable cell lines created from the M14 and SK-MEL-5 cell lines were treated with tetracycline (1 µg/ml) for120 hours. Ras-GTP levels were detected by pull-down assay from 200 mg total protein. Total Ras were detected by immunoblotting of cell lysates. All results in this figure were representative of 3 independent experiments.
Supplementary Table 1: RasGRP3 siRNA information
siRNA-75
siRNA-126
siRNA-774
siRNA1260 / Sense: CAA UGG AGA GCU GGA UAA UAG UUA U
Antisense: AUA ACU AUU AUC CAG CUC UCC AUU G
Sense: CCG AUG GUA UUU AUC UUC CAC UGA A
Antisense: UUC AGU GGA AGA UAA AUA CCA UCG G
Sense: GAC CCA UUC UCA UCU UUC UUC AGA A
Antisense: UUC UGA AGA AAG AUG AGA AUG GGU C
Sense: CAU AAG GAA AUU AGU GGA GUC UGU A
Antisense: UAC AGA CUC CAC UAA UUU CCU UAU G
Supplementary Table 2: Scrambled control and RasGRP3 shRNA sequences
sh236 / 5'-CACCGGTACTGGATTCTGAAGTTTCTCAAGAGGAAACTTCAGAATCCAGTACC-3'
sh854 / 5'-CACCGCAATTACCGCAAGGCCTTTGTCAAGAGCAAAGGCCTTGCGGTAATTGC-3'
Scrambled Control 1 / 5'-CACCGCTGATAGCATTCGACGTCTATCAAGAGTAGACGTCGAATGCTATCAGC-3'
Scrambled Control 2 / 5'-CACCGATAGCATTCGGACGTTGCTATCAAGAGTAGCAACGTCCGAATGCTATC-3'
Supplementary Table 3: Mutations/variants identified in possible cancer genes in the NCI-cancer cell lines described here (Ikediobi et al 2006).
SK-MEL-5 / BRAF V600E
SK-MEL-2 / NRAS Q61R; TP53 G245S
SK-MEL-28 / BRAF V600E; TP53 L145R; EGFR P753S
UACC-62 / BRAF V600E; PTEN P248fsX5
UACC-257 / BRAF V600E
M14 / BRAF V600E; TP53 G266E
MALME-3M / BRAF V600E
LOX-IMVI / BRAF V600E
Supplementary Experimental Procedures
Culture of cell lines
Where indicated, melanocyte culture medium was replaced with melanoma cell culture medium containing RPMI-1640 medium supplemented with 10% fetal bovine serum.
Establishment of the cell lines stably overexpressing wild type and mutated RasGRP3
Human RasGRP3 (KIAA0846) was kindly provided by Dr. J.C. Stone. The coding portion of the sequence was subcloned into the modified pLenti6/V5-D-TOPO® vector by using the pLenti6/V5-D-TOPO® cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. A mutated variant of RasGRP3 (designated sh854m) was also prepared which retained the amino acid sequence of RasGRP3 but which was no longer homologous with shRNA854 (see below). The mutation was introduced using the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) with the following primers: 5'-gttggtctcctccaacggcaattactgtaactacagaaaagc attcgccgactgcgatggcttcaaaatc-3'. Lentivirus containing RasGRP3 or its mutated variant sh854m was generated according to the manufacturer’s instructions. A stable cell line derived from melanocytes overexpressing RasGRP3 fused to a C-terminal V5 tag was created as described before (Pu et al 2005). Studies were carried out on the pooled, stable antibiotic-resistant cells. The control for RasGRP3 overexpressing melanocytes was created by the infection of a positive control lentiviral construct, Lenti6/V5-GW/lacZ, provided by Invitrogen.
siRNA transfection
Stealth RNAi duplexes specific to RasGRP3 (BC027849) were from Invitrogen (Invitrogen, Carlsbad, CA). Sequence information for the Stealth RNAi duplexes used in this study is provided in Supplemental Table 1. Control experiments were performed with a Stealth RNAi Negative Control Duplex pool (containing equal amounts of the low and the medium GC content negative controls) from Invitrogen (Invitrogen, Carlsbad, CA).
Cells were trypsinized and plated on 10 cm tissue culture dishes for 24 h before transfection. Lipofectamine™ 2000 (45 ml) and 1.2 nmol siRNA were applied according to the manufacturer's instructions. Cells were cultured without changing culture medium and the cells were washed and subjected to assays for cell proliferation and apoptosis, to immunoblotting, and to RT-PCR at times between 48 and 120 h after transfection as indicated.
Cell proliferation assay
Cell proliferation was measured using the CyQuant NF cell proliferation assay as described by the manufacturer (Invitrogen, Carlsbad, CA). The fluorescent signals were quantified on an Infinite M200 fluorescent microplate reader (Tecan, Durham, NC) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
For measurement of cell growth using the IncuCyteÔ (Essen Instruments Inc., Ann Harbor, MI), the control melanocytes and RasGRP3-melanocytes (1.0 x 104 cells/well) were plated on 24 well plates. Phase contrast images of cells were taken in the IncuCyte™ at one hour intervals for 60 hours. The data were analyzed by the IncuCyte™ cell proliferation assay software.
Detection of RasGRP3 by reverse transcriptase PCR (RT-PCR)
Total RNA was purified from cells using TRIzol reagent (Invitrogen, Carlsbad, CA). Two sets of published RasGRP3 primers were used for the PCR (Stope et al 2004, Yamashita et al 2000). RT-PCR reactions were carried out using the SuperScript III one-step RT-PCR system (Invitrogen), 1 μg total RNA and RasGRP3 gene specific primers (200 nM). RT-PCR products were analyzed on 1% agarose gels. The identity of the bands was confirmed by purification using the MinElute Gel extraction Kit (Qiagen, Valencia, CA) followed by DNA sequencing (DNA Minicore Facility, Center for Cancer Research, NCI).
Probes for the RasGRP3 in situ hybridization (ISH)
Two published probes − probe 1 and probe 2 − were generated by cloning 327bp and 205bp long cDNA fragments of the RasGRP3 (GenBank accession number GI:24762238) into a Bluescript II SK vector (Stratagene, La Jolla, CA) (Roberts et al 2004, Yamashita et al 2000). The identity of the clones was confirmed by DNA sequence analysis. [35S]UTP -labeled (Per-Form Hungaria Kft, Budapest, Hungary) riboprobes were prepared by in vitro transcription using a MaxiScript Kit (Ambion, Austin, TX) with T3 and T7 RNA polymerases in antisense and sense directions, respectively.
In situ hybridization histochemistry
Slides containing paraffin embedded human multiple melanoma tissue arrays (US Biomax Inc, Rockville, MD) were heated at 65ºC for 1 hr and deparaffinized. They were rinsed once in physiological saline and washed for 2 x 5 min in PBS (pH 7.2). All the solutions were sterile and made with DEPC treated autoclaved water. Antigen retrieval was performed by microwave treatment keeping the slides at boiling temperature in 0.1 M citric acid (pH 6.0) for 2 x 5 min. After cooling down to room temperature, the slides were incubated in 100 µg/ml freshly prepared pepsin (Sigma) solution at 37ºC for 15 min. Next, the slides were rinsed with sterile water, washed in PBS for 3 x 5 min and incubated in 0.1 M triethanolamine-HCl buffer (pH 8.0) containing 0.25% of acetic anhydride (all from Sigma). After a quick rinse in 2 x SSC buffer (pH 7.0), the slides were dehydrated in a graded ethanol series (70%-85%-95%-100%-95%) and air dried. Tissue arrays were divided into four groups and hybridized overnight at 55ºC with 1.5 x 106 cpm /slide [35S] UTP -labeled probe 1 or 2, either using the sense or the antisense strand. Next day the arrays were washed in 4 x SSC/1 mM DTT for 4 x 5 min, then in 20 µg/ml RNase A (Sigma) at 37ºC for 30 min. After 5 min washes in 2 x SSC/1 mM DTT, 1 x SSC/1 mM DTT, and 0.5 x SSC/1 mM DTT at room temperature, slides were placed two times for 40 min at 65°C in 0.1 x SSC/1 mM DTT. Slides were then dehydrated, air dried, and apposed to a BAS-MS 2340 imaging plate (Fuji Photo Film Co., LTD, Kanagawa, Japan) for 5 days. Data were read out by a phosphorimager (FLA-3000, Fuji ), and the slides were dipped into NTB nuclear track emulsion (Eastman Kodak Co., Rochester, NY, USA) for 3 weeks and kept at 4ºC in the dark. The emulsion-coated slides were developed using Kodak Dektol developer and fixer at 18ºC (Kodak). Tissues were counterstained with 0.5% Giemsa solution (Sigma), air dried and coverslipped using Cytoseal 60 mounting medium (Stephens Scientific, Riverdale, NJ). Pictures were taken with a BX51 microscope (Olympus Hungary Kft, Budapest, Hungary) equipped with a QICAM 10 bit- mono camera with RGB slider (QImaging, Surrey, BC, Canada).
Analysis of Ras activation using the Raf1-RBD-GST pull-down assay
After being collected and washed once with Dulbecco’s phosphate buffered saline, cells were lysed by addition of cold lysis buffer (which consisted of binding buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, 2.5 mM MgCl2, 10% glycerol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 1 mM aprotinin) together with 0.5% NP-40) followed by incubation at room temperature for 5 min. Lysates were clarified by centrifugation at 14,000 x g for 5 min at 4 °C. Activation of Ras from 200 mg total protein was evaluated using the procedure described by Teixeira and coworkers (Teixeira et al 2003).
Anchorage-independent growth assay in soft agar
The soft agar experiments were performed in six well plates. The 1 ml bottom layer contained 0.6% of SeaPlaque Agarose (Lonza Inc, Riverside, PA) in complete medium with (three bottom wells) or without (three upper wells) 1 µg/ml tetracycline and the 1 ml top layer contained 0.3 % SeaPlaque agarose in complete medium with (three bottom wells) or without (three upper wells) 1 mg/ml tetracycline and 10,000 cells. The plates were cultured for 10 days at 37oC with 5 % CO2. Multiple images were taken from the lower half of each well (about 10 images) with a 2X objective on the microscope and a digital camera. The number of colonies greater than the median size of the non-treated control was determined using Image J software (National Institutes of Health (NIH) Image).
Quantitative Real-Time PCR (Q-PCR) from melanoma tissue cDNA array
qPCR analysis of different melanoma cDNAs was performed on TissueScan Melanoma Tissue qPCR Array Panel I (OriGene, Rockville, MD). This array contained normalized amounts of cDNA (2-3 ng of cDNA normalized to beta-actin) from different stage III, IIIA, IIIB, IIIC and stage IV tumors and three normal skin controls. The expression of RasGRP3 cDNA was determined on a Bio-Rad iCycler iQ from triplicate samples using iQ SYBR Green Supermix (Bio-Rad) with previously validated RasGRP3 primers (Roose et al 2005)and the control beta actin primers (single experiment) provided by Origene (Rockville, MD). Relative quantitation was calculated using the 2–(ΔDCt) formula, where ΔDCt represents the cycle difference between RasGRP3 and beta actin used as internal control. The data are presented as fold change in gene expression normalized to beta actin and relative to the average of the three normal skin tissues.
Imaging of cultured cells
Living cells were imaged using a Nikon Eclipse TE300 Inverted Microscope (Nikon, Japan).
Statistical Analysis
To assess the significance in the differences in cell proliferation, anchorage independent growth and xenograft studies, a Student’s two-tailed t test was performed. The minimal level of significance was p < 0.05.