Tissue Distribution Analysis—PCR primer pairs were designed for both alternative first exons of GLI1. Multiple tissue cDNA panels from BD Biosciences were used with the primers obtained from MWG-Biotech (Ebersberg, Germany). Each reaction consisted of 1× ThermoPol Reaction buffer (New England Biolabs, MA), 0.2 mm of each dNTP, 1.0 μm forward primer for the alternative first exons (5′-GAGCCCAGCGCCCAGACAGA for exon 1 or 5′-CTGTCTCAGGGAACCGTGGGTCTTTGT for exon 1A), 1.0 μm reverse primer for exon 4 (5′-GGCATCCGACAGAGGTGAGATGGAC), 0.05 units/μl of Taq DNA polymerase (New England Biolabs), and 1 ng of cDNA in a total volume of 25 μl. Thirty-five cycles with 20 s at 94 °C, 20 s at 66 °C, and 30 s at 72 °C were performed on a PTC-200 Peltier Thermal Cycler (MJ Research, MA). Amplifications without exogenous cDNA were used in all sets of experiments as a negative control. The PCR products were analyzed on a 4% NuSieve 3:1 agarose gel (FMC BioProducts, ME). All DNA bands were sequence verified by using BigDye Terminator version 1.1 Cycle Sequencing Kits and an ABI prism DNA sequencer (Applied Biosystems, CA).
cDNA Expression Constructs—For construction of the GLI1ΔN expression plasmid, we first performed a PCR amplification using the Expand PCR System (Roche Diagnostics) on cDNA from HEK293 cells, which express this variant, and a GLI1 primer set (5′-CTCAAGCTTGGCACCATGAGCCCAT and 5′-CACAGATTCAGGCTCACGCTTC). The PCR product and a full-length 3′-FLAG-tagged GLI1 expression plasmid in pCMV5 were digested with HindIII and XhoI restriction enzymes and then ligated resulting in a construct with deleted exons 2 and 3. Additionally, SalI- and BglII-digested FLAG-tagged GLI1FL or GLI1ΔN fragments were subcloned into SalI- and BamHI-digested pBABEpuro. The 3′-GFP-tagged GLI1FL and GLI1ΔN constructs were generated from the HindIII and NdeI fragments of FLAG-tagged GLI1FL/GLI1ΔN expression plasmids in pCMV5, the NdeI and NotI fragments of EGFP-tagged GLI1 (27 (link)), and the HindIII and NotI fragments of pEGFP-N3 (BD Biosciences). All constructs were verified by sequencing.
The other expression and reporter constructs used have been described in previous reports, the full-length 3′-FLAG-tagged PTCH1 constructs with the alternative first exons, 1, 1B, and 1C (12 (link)), 5′-Myc-tagged PTCH1 with the alternative first exon 1B (28 (link)), PTCH2 (13 (link)), Dyrk1WT, Dyrk1KR (27 (link)), and the SUFU expression constructs (29 (link)), and the 12xGLIBS-luc (29 (link)), PTCH2-luc (13 (link)), IL1R2-luc (30 (link)), and a mouse Ptch1-1B-luc5 reporter construct.
Cell Culture and Transfection—The human embryonic kidney cell line HEK293 and the murine fibroblast cell lines NIH3T3 and C3H10T1/2 were cultured as described before (12 (link), 13 (link)). Human fibroblast cell line hTERT-BJ1 (BD Biosciences), medulloblastoma cell line Daoy, which stably expresses EGFP (EGFP-Daoy), rhabdomyosarcoma cell lines RMS13 and CCA, prostate carcinoma cell lines DU145 and 22Rv1, pancreas carcinoma cell line Panc1, gastric adenocarcinoma cell line AGS, lung adenocarcinoma cell lines H22 and H522, and lung carcinoma cell line A549 were cultured according to the ATCC-LGC Promochem (Middlesex, United Kingdom) and Metalab (Bologna, Italy) recommendations. Expression constructs for the GLI1 splice variants and other pathway components, and the appropriate reporter construct were transfected into cultured cells using FuGENE 6 (Roche Diagnostics). For selection, NIH3T3 and C3H10T1/2 cells were co-transfected with the neomycin-resistant pcDNA3.1His (Invitrogen) and then incubated with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum with 0.6 mg/ml G418 (Sigma).
Reporter Assays—Reporter assays were performed as described previously (12 (link), 13 (link)). 12xGLIBS-luc, Ptch-1B-luc, PTCH2-luc, or IL1R2-luc reporter constructs together with the internal control, pRL-SV (Promega) were co-transfected into the appropriate cell lines. Normalized luciferase activity was determined with the dual-luciferase reporter assay system (Promega) using the Luminoskan Ascent (Thermo Electron Corporation, MA) according to the supplier's recommendations. All experiments were analyzed independently three times.
Immunofluorescence Microscopy—Transfected NIH3T3 cells were plated into the Lab-tec chamber slide (Nalge Nunc International, NY). The next day, the cells were rinsed with phosphate-buffered saline and fixed in 4% paraformaldehyde for 15 min and cold methanol for 10 min. Nonspecific binding of the primary antibodies was reduced by first blocking the cells using a solution of phosphate-buffered saline with 5% normal goat serum for 60 min. Then anti-FLAG M2 mouse monoclonal (1:800, Sigma), anti-hemagglutinin rabbit polyclonal (Y-11, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-SUFU rabbit monoclonal (C81H7, 1:200, Cell Signaling Technology), and anti-PTCH1 rabbit polyclonal (H-267, 1:100, Santa Cruz Biotechnology) in 0.3% Triton X-100/phosphate-buffered saline were added and incubated overnight at 4 °C. After the cells were washed, fluorescent-tagged secondary antibodies, Alexa Fluor 488-goat anti-mouse IgG (1:6000, Invitrogen), and Alexa Fluor 546-goat anti-rabbit IgG (1:6000, Invitrogen) were applied. Three phosphate-buffered saline washes (5 min) were also used after each treatment. The nuclei were stained with 5 μm DRAQ5 (Alexis Biochemicals, Lausen, Switzerland). Slides were mounted in FluorSave reagent (Carbiochem, Darmstadt, Germany). Fluorescence images were collected using a LSM510 (Carl Zeiss, Oberkochen, Germany) confocal laser-scanning microscope with a Plan APOCHROMAT ×63/1.4 OilDIC objective lens.
Western Blotting—48 h after transfection, proteins were extracted by lysis buffer (50 mm Tris (pH 7.4), 1%. SDS, 250 mm NaCl, 2 mm dithiothreitol, 0.5% Nonidet P-40, 1% phosphatase inhibitor mixture 1 (Sigma), and 1% mammalian protease inhibitor mixture (Sigma)). After normalization based on protein concentration measured by the DC Protein assay kit (Bio-Rad), samples were run on a SDS-acrylamide gel. Thereafter, proteins were transferred onto Hybond-ECL nitrocellulose membrane (GE Healthcare). GLI1 isoforms or α/β-tubulin as internal control were determined by the use of anti-GLI1 rabbit polyclonal (Cell Signaling Technology) or α/β-tubulin rabbit polyclonal antibodies (Cell Signaling Technology). Horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare) was used as the secondary antibody, followed by detection of these proteins with the Western Lightning Western blot Chemiluminescence Reagent Plus (PerkinElmer Life Sciences).
RNA Isolation and Reverse Transcription—Total RNA was isolated from cultured cells, using the RNA-Bee reagent (Tel-Test Inc., TX) or the RNeasy kit (Qiagen GmbH, Hilden, Germany) following the manufacturer's protocol. For reverse transcription, 5 μg of total RNA, 4 μl of a 2.5 mm dNTP mixture (2.5 mm each), and 0.5 ng of oligo(dT)18(A/C/G)(A/C/G/T) primer in a total volume of 12 μl were denatured at 65 °C for 5 min. After cooling the mixture on ice, 4 μl of 5× RT buffer, 2 μl of 0.1m dithiothreitol, and 1 μl of ribonuclease inhibitor (New England Biolabs) were added. Following incubation at 45 °C for 2 min, 1 μl of SuperScript II RT (Invitrogen) or water (negative control) was added. Then the mixture was incubated at 45 °C for 90 min. The reaction was stopped by heat inactivation at 75 °C for 15 min.
Real-time RT-PCR—Relative levels of the GLI1 variant RNAs or transcripts of the pathway target genes Gli1, Ptch1, Ptch2, and Sfrp were quantified by real-time RT-PCR using SYBR Green. Duplicate samples of each PCR mixture, each containing 4.7 μl of POWER SYBR Green PCR master mixture (Applied Biosystems), 0.3 μl of a 10 pmol/μl of primer mixture, 0.3–1.0 μl of cDNA, and water to a total volume of 10 μl were transferred into a 96-well plate on an ABI 7500 Fast Real Time PCR System (Applied Biosystems). The samples were initially incubated at 95 °C for 3 min, followed by 45 cycles with 95 °C for 15 s, 65 °C for 15 s, and 72 °C for 30 s. Dissociation curves were generated after each PCR run to ensure that a single, specific product was amplified. The results were analyzed with the comparative Cycle threshold (Ct) method. For normalization, we used the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB), hypoxanthine phosphoribosyltransferase 1, ribosomal protein, large, P0 (RPLP0), and TATA box-binding protein for the human samples and Acidic ribosomal protein (Arp), Hprt1, and Gapdh for the mouse samples. From the results obtained the two housekeeping genes with the most scattered values were eliminated and then all data were normalized by the average Ct of the remaining housekeeping genes. Samples that were not reverse transcribed were used as negative controls. The PCR primers are shown in supplemental Table S1. The primer specificity was verified by BLAST searches and analysis of the PCR products by agarose gel electrophoresis followed by sequence determination.
Polysome Analysis—GLI1 transcripts associated with polysomes were fractionated as described (31 ). In brief, cytoplasmic extracts from 1 × 107 RMS13 cells lysed in the presence of 50 μg/ml of cycloheximide or 25 mm EDTA were layered over a 15–40% sucrose (w/v) linear gradient and centrifuged in a SW55 Ti rotor (Beckman Coulter) for 120 min at 45,000 × g at 4 °C. Twenty fractions were collected and the RNA was isolated using the RNA-Bee reagent and LiCl precipitation. Transcript levels were measured by real-time RT-PCR as described above but with the use of random primer 6, 5′(dN6) (New England BioLabs). The percentage of individual transcripts in each gradient fraction was calculated from the Ct values. The isolated RNA from each fraction was also analyzed using denaturing 1.2% formaldehyde-agarose gel electrophoresis.
Statistical Analysis—The statistical significance was calculated using the PRISM software (GraphPad Software, CA).