Calpain as an effector of the Gq signaling pathway for inhibition of Wnt/beta -catenin-regulated cell proliferation -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Li and Iyengar (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.202355799

Supporting Materials and Methods
Cell Lines, Plasmids, and Reagents.
HEK 293 cells (ATCC CRL-1573) and SW480 cells (ATCC CCL-228) were cultured in DMEM, supplemented with 10% FBS.

WT and mutant Ga subunits and M2R and M3R used in this study were purchased from GUTHRIE cDNA Resource Center (Sayre, PA). For DN-XTCF3VP16 construct, DN-XTCF3 was amplified by PCR and subcloned into pcDNA3.1 (Invitrogen) with its C terminus fused to the transactivation domain of VP16 that was amplified by PCR from pTet-On (CLONTECH). The construct was verified by DNA sequencing.

To enrich transfected cells, Q209L-Gaq cDNA was subcloned into pIRES2-EGFP (CLONTECH). SW480 cells transfected with pIRES2-EGFP or pQ209L-Gaq-IRES2-EGFP with FuGENE 6 (Roche Diagnostics) were sorted out for EGFP expression by fluorescence-activated cell sorting 24 h after transfection.

For stable expression, cDNAs for M3R and DN-XTCF3VP16 were subcloned into retroviral vectors pMSCVpuro and pMSCVneo (CLONTECH), respectively. SW480 cells were infected with M3R or DN-XTCF3VP16 retrovirus packaged by GP2-293 cells (CLONTECH). Cells stably expressing M3R or DN-XTCF3VP16 were selected by puromycin (2 mg/ml) or G418 (500 mg/ml).

The following inhibitors were purchased from Calbiochem: PLCb inhibitor (U-73122), PKC inhibitors (BIM-I and calphostin C), calpain inhibitors (ALLN, ALLM, E-64, calpeptin, and calpastatin), proteasome inhibitor (lactacystin), proteasome and calpain inhibitor (MG-132), cell permeable calcium chelator (BAPTA/AM), Ca²⁺-ATPase inhibitor (thapsigargin), and protein synthesis inhibitor (cycloheximide).

TOPFLASH/FOPFLASH Reporter Assay.
SW480 cells were cultured on 12-well plates for 24 h before transfection. Cells were transfected in duplicatewith 0.25 µg of pTOPFLASH or pFOPFLASH, 5 ng of pcDNA3.1LacZ/MycHis (Invitrogen), and the other required constructswith FuGENE 6 (Roche Diagnostics). Thetotal amount of DNA was 0.5 µg for each sample, which was keptconstant by adding empty plasmid DNA if necessary. Forty-eight h after transfection, cells were lysed with 1´ passive lysis buffer (Promega). Lysate was assayed for luciferase and b-galactosidase activities, by using a luciferase assay system (Promega) and luminescent b-galactosidase detection kit II (CLONTECH) according to the manufacturer’s instructions. Luminescence was measured by using a TD-20/20 luminometer (Turner Design, Sunnyvale, CA). Luciferase activity was normalized for transfection efficiency by b-galactosidase activity. Relative TCF/LEF activity is defined as the ratio of pTOPFLASH to pFOPFLASH. The ratio for control is usually defined as 1. Whole-Cell Extract, Cell Fractionation, and Immunoblotting.
Cells were washed twice with ice-cold PBS and lysed in buffer (50 mM Tris× HCl , pH8 .0/120mM NaCl/0.5% NP-40) supplemented with 0.01 vol of protease cocktail (Sigma). Lysateswere cleared by centrifugation at 12,000 g for 20 minat 4°C.

For cell fractionation, cells were washed, harvested with ice-cold PBS, and centrifuged at 450 g for 5min at 4°C. Pellet was suspended with twice volume of ice-cold low salt buffer (10 mM Hepes, pH 7.9/1.5 mM MgCl₂/10 mM KCl/0.05% NP-40) supplemented with protease inhibitor cocktail (Sigma) and incubated on ice for 10 min. Nuclei were pelleted at 10,000 g for 1 min. Supernatants were cleared by centrifugation at 100,000 g for 45 min and used as cytosolic fractions. Nuclei were extracted with twice volume of ice-cold high salt buffer [20 mM Hepes, pH 7.9/1.5 mM MgCl₂/0.42 M NaCl/0.2 mM EDTA/25% (vol/vol) glycerol] supplemented with protease inhibitor cocktail (Sigma) by rotating at 4°C for 30 min. Nuclear extract was cleared by centrifugation at 10,000 g for 10 min.

Protein concentrations were then determined by usingthe BCA protein assay kit (Pierce). A 10- to 40-µg aliquotof protein per sample was subjected to SDS/PAGEand transferred to Hybond-C extra nitrocellulose membrane (Amersham Life Science) followed by immunoblotting and ECL detection. The following antibodies were used: mouse anti-cyclin D1 (1:100; Santa Cruz Biotechnology), mouse anti-b-actin (1:25,000; Sigma), mouse anti-b-catenin (specific for C terminus; 1:5,000; Transduction Laboratories, Lexington, KY), mouse anti-b-catenin (specific for N terminus; 1:1,000; Upstate Biotechnology), and horseradish peroxidase-conjugated goat anti-mouse IgG (1:2,500; Pierce).

Immunofluorescence Microscopy.
SW480 cells were grown on glass cover slips. Transfected cells were collected for analysis after 24–48 h. For the staining, cells were fixed and permeabilized with 4% paraformaldehyde (freshly prepared, in PBS) and 0.1% Triton X-100 for 15 min, and blocked with 1% BSA for 30 min, and subsequently incubated at room temperature with primary and secondary antibodies for 60 min each. The following antibodies were used for the immunostaining: FITC-conjugated mouse anti-b-catenin (1:250; Transduction Laboratories), rabbit anti-b-catenin (C terminus) (1:1,000; Sigma), mouse anti-m-calpain (1:250; Calbiochem), mouse anti-m-calpain(1:50; Calbiochem), Alexa Fluor 488 goat anti-mouse IgG (1:500; Molecular Probes), and Alexa Fluor 568 goat anti-rabbit IgG (1:500; Molecular Probes). Images were collected on a Leica TCS-SP (UV) confocal microscope in the Mount Sinai Microscopy Facility.