finally 20% sucrose solution at 4uC for 12 h, respectively. They were embedded in OCT compound and frozen. Thin sections were prepared with a cryostat (Leica). Cultured ureteric bud cells and MDCK cells were fixed in 4% PFA for 10min. Samples were incubated with the primary antibodies listed in Table S1. Signals were visualized with Alexa Fluor-conjugated secondary antibodies (Molecular Probes) used at a dilution of 1:500. F-actin bundles were detected with Alexa Fluor 488- or 555-conjugated phalloidin (Molecular Probes) at a dilution of 1:250. Nuclei were counter- stained with Hoechst 33342 (Sigma). Flow cytometric analysis and cell sorting Kidneys isolated from embryos were incubated with 0.25% trypsin and 0.5mM EDTA at 37uC for 30min and dissociated into a single-cell suspension. Cells were co-stained with fluorescein- and biotin-conjugated antibodies, washed, incubated with allophyco- cyanin-conjugated streptavidin (Invitrogen), and analyzed by FACSCalibur (Becton Dickinson). Dead cells were excluded by propidium iodide staining. For cell sorting, MoFlo XDP (Beckman Coulter) was used. RNA extraction and quantitative reverse transcription PCR (RT-PCR) Total RNA was extracted from each cell preparation using a First Pure RNA kit (Takara). Total RNA (0.3 mg) and random hexamer primers were used to synthesize cDNA with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative Real- time RT-PCR experiments were conducted with a LightCycler (Roche Diagnostics) and SYBR Premix Ex Taq (Takara-bio). Primers for Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were used as the control. The pairs of primers used were as follows: 59-TGAACGGGAAGTCACTGG-39 (Gapdh primer, sense), 59- TCCACCACCCTGTTGCTGTA-39 (Gapdh primer, antisense), 59-CTGACCTAGACTCCGAGCTG-39 (TROP2 primer, sense), 59-CGGCCCATGAACAGTGACTC-39 (TROP2 primer, anti- sense), 59-AGGGGCGATCCAGAACAACG-39 (EpCAM primer, sense), 59-ATGGTCGTAGGGGCTTTCT-39 (EpCAM primer, antisense), 59-AAGTACAGCACCAAGTTCCTCAGC-39 (Wnt9b primer, sense), 59-GAACAGCACAGGAGCCTGACAC-39 (Wnt9b primer, antisense), 59-CTGAATCAGACGCAACACTG- TAAAC-39 (Wnt11 primer, sense), 59-CTCTCTCCAGGTCAAG- CAGGTAG-39 (Wnt11 primer, antisense), 59-GGAAGGT- GTCGTTGATGAAGGA-39 (Ret primer, sense), 59-CTCAGCA- TCCGCAATGGTG-39 (Ret primer, antisense), 59-CACTCCTG- GATTTGCTGATGT-39 (GFRa primer, sense), 59-CTGAAG- TTGGTTTCCTTGCCC-39 (GFRa primer, antisense), 59-CCC- GATGACCAAATGAAAGACG-39 (Sall1 primer, sense), 59-TA- GAGAGGTTGTGATCGCTGA-39 (Sall1 primer, antisense). Culture of ureteric bud cells and transfections Ureteric bud cells from E14.5 kidneys were suspended in DMEM/F12 (1:1) medium (Gibco) containing 10% FBS (JRH) and antibiotics. Cells were seeded on type-I collagen-coated dishes. Relative cell size was measured as the actin-positive area using ImageJ (Rasband, W.S., NIH) and cellular area was determined. To express mouse TROP2, its cDNA fragment was inserted upstream of IRES-GFP in the vector pTIB731, provided by Dr. Tohru Itoh (University of Tokyo). Ureteric bud cells were sorted and cultured for 24 h prior to transfection. The transfection was carried out using polyethyleneimine ‘‘Max’’ reagent (Polysciences Inc.). Twenty four hours later, the cells were immunostained and the size of the GFP+ transfected cells was examined under a fluorescence microscope. TROP2 Defines Branch Formation in Fetal Kidney MDCK cell culture and virus infection MDCK cells were cultured in DMEM (Gibco) containing 10% FBS (Equitech) and antibiotics [34]. For expression of mouse TROP2, its cDNA was inserted into the pMxs vector. The pMxs- IRES-GFP vector was used as a negative control. Retrovirus was produced using the retrovirus packaging cell line PLAT-A and MDCK cells were infected as described previously [47]. Cells expressing TROP2 or GFP were sorted using anti-TROP2 Abs or GFP and MDCK cells expressing GFP (MDCK-control) or TROP2 (MDCK-TROP2) were established. Immunoblotting MDCK cells were lysed in TNE buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM Pefabloc SC (Roche Applied Science), and 10 mg/ml leupeptin), and the whole cell extracts were separated by SDS-PAGE and transferred onto polyvinyli- dene difluoride membranes (Immobilon-P; Millipore). The membranes were subjected to Western blot analyses with anti- TROP2, anti-Fak or anti-p-Fak (Tyr397) antibody. Anti-b-actin antibody was used to confirm equal loadings. Cell spreading assay MDCK cells were detached by trypsin and resuspended in serum-free medium at 56103 cells/ml. The cell suspension (1 ml) was added to type I collagen-coated coverslips. Cells were allowed to spread, and spread cells were scored manually under a phase- contrast microscope at 1 and 2.5 h after plating. Spread cells were defined as the cells with extended processes. Wound healing assay MDCK cells were grown on collagen-coated 60 mm dishes. The monolayer of cells was scratched with a pipette tip and the culture medium was changed to serum-free medium. Pictures were taken at 0, 6 and 16 h and the length of the gap between cell sheets was measured at 0 and 6 h. Collagen gel culture MDCK cells were detached from plates with trypsin and triturated into a single cell suspension. Cells were diluted to 0.56103 cells/mL in a type I collagen solution and the cell solution was plated in a 24-well plate (500 mL/well). After incubation at 37uC to allow the collagen solution to gel, 1 mL of the culture medium was added. The medium was changed every 24 h, and after 2 days, medium containing 20 ng/mL of HGF was added and cultured for additional 2 days. Supporting Information Figure S1 (TIF) Figure S2 kidney. (TIF) Figure S3 kidney. (TIF) Figure S4 TROP2. (TIF) Figure S5 (TIF) Expression of TROP2 and Ki67 in E14.5 kidney. Expression of Claudin7 and TROP2 in E14.5 Expression of collagen-I and EpCAM in E14.5 Establishment of MDCK cells expressing Expression of laminin in E14.5 kidney. PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28607