Supplement 2002-06144A

Cell lines, antibodies and immunoblotting.

C57MG cells were cultured in DMEM, 10% FBS, 10 g/ml insulin and antibiotics. Wnt3A specific antisera were obtained by immunizing rabbits with a GST fusion protein containing amino acids Ser 220 to Asn 279 of Wnt3A. The sera were affinity purified against the immunogen. For immunoblotting samples were resolved on SDS-10% polyacrylamide gels, and transferred to nitrocellulose. The membranes were incubated for 1 hour in blocking solution (1% BSA, 3% nonfat dry milk in Tris buffered saline containing 0.2% Tween 20) and then overnight in blocking solution containing either rabbit anti-Wnt3A (1:2000) or mouse anti--catenin (1:500, Santa Cruz Biotech). Proteins were detected using HRP conjugated secondary antibodies (Santa Cruz Biotech) with the ECL western blot detection reagents (Amersham Biosciences).

Target gene expression in Xenopus animal cap explants.

Animal caps were explanted from early- to mid-blastulae and cultured in 1X Modified Barths Saline with either Wnt3A protein (100 to 400 ng/ml) or an equivalent amount of control buffer. Explants were harvested at early gastrula stage and homogenized in Trizol (Invitrogen). Total RNA purified and used for PCR amplification.

Morphological transformation of C57MG cells.

C57MG cells were not treated or treated with 100 ng/ml Wnt3A in DMEM, 10% FBS, 10 g/ml insulin and antibiotics for two days. The medium was then replaced with IS-CHO (Irvine Scientific) -/+ 100ng/ml Wnt3A and photos were taken two days later.

Hematopoietic stem cell proliferation and transplantation assays

Mice

BCL-2 transgenic mice30 were used at 6-10 weeks of age. Mice were bred and maintained on acidified water in the animal care facility at Duke University Medical Center.

HSC isolation

HSCs were sorted from mouse bone marrow as described (ref. 19 and references within). All cell sorting and FACS analysis was carried out on a FACSVantage (Becton Dickinson) at the Duke Cancer Center FACS facility. Cells were sorted based on expression of c-kit, Sca-1, low levels of Thy1.1, and low to negative levels of lineage markers (Lin).

In vitro HSC proliferation assays

Freshly purified HSCs were plated at one-ten cell s per well in Terasaki plates and cultured in X-vivo 15 medium (BioWhittaker), 10% FBS, 5x10-5M 2-Mercaptoethanol, and 1x10-4M random methylated beta-cyclodextrin (CTD, Inc.) with the indicated growth factors. Single cells were stimulated with SLF (7.5 ng/ml) in the presence or absence of purified Wnt3a (~100 ng/ml). Proliferation was monitored by counting the number of cells in each well at defined intervals. Cells cultured in bulk (3500 cells per well in 96-well plates) for phenotypoic analysis were treated with or without purified Wnt3a (~100 ng/ml) in X-vivo 15 medium (BioWhittaker), 10% FBS, 5x10-5M 2-Mercaptoethanol, and 1x10-4M random methylated beta-cyclodextrin (CTD, Inc.).

In vivo analysis of HSC function

HSCs were cultured in vitro in the presence of Wnt3A and retro-orbitally injected into groups of 4-6 congenic recipient mice irradiated with 9.5 Gy using a 200kV x-ray machine, along with 300,000 rescuing Sca-1neg host bone marrow cells. Transplanted mice were bled at regular periods to determine the percent of the hematopoietic compartment contributed by the donor cells. Donor and host cells were distinguished by allelic expression of CD45 (Ly5) or expression of the BCL2 transgene.