Adherent BMEL cell culture
BMEL 9A1 cells, kindly provided by M. Weiss , were grown in RPMI 1640 (Invitrogen, Cergy-Potoise, France) supplemented with 10% foetal calf serum (Eurobio, Les Ulis, France), 50 ng/ml Epidermal Growth Factor (PeproTech, Levallois Perret, France), 30 ng/ml Insulin-like Growth Factor II (PeproTech), and 10 μg/ml human insulin (Roche, Meylan, France). Cells were cultured on dishes coated with Collagen I (Becton-Dickinson, Le Pont de Claix, France) in a humidified 5% CO2 atmosphere at 37°C. Cells were passaged every 3 to 4 days at 70.103 cells for a 100 mm dish, and never let sit at confluence.
Aggregate BMEL cell culture
1,5.103 cells per well were seeded onto 96-well plates coated with Collagen I and cultured for two days as indicated previously. Cells were then dissociated with trypsin-EDTA (Eurobio) and 1.104 cells per well were seeded into 96-wells coated with poly(2-hydroxyethyl-methacrylate) (Sigma-Aldrich, Saint Quentin Fallavier, France) to which cells do not attach, but form floating aggregates within 24 hours. Aggregates were collected for analysis 4 days after seeding.
mRNA isolation and RT-PCR reactions
mRNA was isolated with the Dynabeads mRNA Direct Kit (Invitrogen). First-strand cDNA was synthesized using M-MuLV reverse transcriptase (Promega, Charbonnières, France) and 400 nM random pentadecamer primers (Eurogentec, Liège, Belgique) . cDNA was amplified by PCR over 35 cycles [94°C for 30 sec, 59 or 60°C for 30 sec, and 72°C for 40 sec] with RedTaq DNA Polymerase (Sigma-Aldrich) on a 9700 thermocycler (Applied Biosystems, Courtaboeuf, France) using the following primer pairs: β-actin (forward) 5'-AGCCATGTACGTAGCCATCC-3' (reverse) 5'-CTCTCAGCTGTGGTGGTGAA-3'; hnf6 (forward) 5'-CTGTGAAACTCCCCCAGGTA-3' (reverse) 5'-TCATCCCGCATAAGTGTGAA-3'; ngn3 (forward) 5'-GAGTTGGCACTCAGCAAACA-3' (reverse) 5'-TCTGAGTCAGTGCCCAGATG-3'; pdx1 (forward) 5'-CTGCGAGCTTCTGGAAAAAC-3' (reverse) 5'-CTGCTGGTCCGTATTGGAAC-3'; neuroD (forward) 5'-GAAAGCCCCCTAACTGACTGC-3' (reverse) 5'-GCACTTTGCAGCAATCTTAGCAAAA-3'; nkx2.2 (forward) 5'-GGTGGAGCGATTGGATAAGA-3' (reverse) 5'-TGCCATCAACCTTTTCATCA-3'; nkx6.1 (forward) 5'-AGTGATGCAGAGTCCGCCG (reverse) TCCTCATTCTCCGAAGTC-3'; hes1 (forward) 5'-CCTCTGAGCACAGAAAGTCATC-3' (reverse) 5'-TCCAGAATGTCTGCCTTCTC-3'; insulin1 (forward) 5'-CATCAGCAAGCAGGTYATTG-3' (reverse) 5'-CACTTGTGGGTCCTCCACTT-3'; insulin2 (forward) 5'-AGGACCCACAAGTGGCACA-3' (reverse) 5'-GAGGGGTAGGCTGGGTAGTG-3'; ptf1a (forward) 5'-GTAACCAGGCCCAGAAGGT-3' (reverse) 5'-CCTCTGGGGTCCACACTTTA-3'; amylase (forward) 5'-GGCCTTCTGGATCTTGCAC-3' (reverse) 5'-TCCTTGGGAGAACCATTTTG-3'.
Quantitative real-time RT-PCR was performed on 1 μl of cDNA with Prism 5700 sequence-detection system (Applied Biosystems). Cycling parameters were initiation at 50°C for 2 min and denaturation at 95°C for 10 min followed by 40 cycles of denaturation 95°C for 15 sec and annealing at 60°C for 1 min. Mouse hes1 (D16464) and β-actin (NM_007393) expression were amplified using 300 nM hes1 (forward) CCTCTGAGCACAGAAAGTCATC (reverse) GCATCCAAAATCAGTGTTTTCA, and β-actin (forward) CTCTTCCAGCCTTCCTTCCT (reverse) GGGCAGTGATCTCTTTCTGC primers in Power Sybr Green Master Mixture (Applied Biosystems), Relative real time PCR data were processed according to a standard curve based method  generating reference gene normalized expression values. Statistical comparisons between groups of treated versus untreated samples were performed using a non parametric Mann-Withney test.
Ngn3, Pdx1, and Hes1 immunostaining was carried out on cytospins of 40,000 BMEL cells after 4 days of adherent or suspension culture. Cells were fixed in 4% paraformaldehyde, permeabilized in PBS-Triton X-100 0,2%, and blocked in BPS, 1%BSA, 10% goat serum before incubation with primary antibodies in PBS -1% BSA. Primary antibodies used were: rabbit anti-Pdx1(1:3000), a gift from C. Wright, rabbit anti-Ngn3 (1:3000), a gift from M. German, and rabbit anti-Hes1 (1:60), a gift from T. Sudo. Secondary biotinylated goat anti-rabbit Ig antibody (Dako E0432) was used at 1:300 and detected with streptavidine-conjugated HRP (Dako, P397) and DAB substrate (Dako, SK-4100).
After harvest with trypsin-EDTA, cells were washed with D-PBS and lysed in RIPA lysis buffer (Upstate, Euromedex, Mundolsheim, France) supplemented with protease inhibitors (Sigma-Aldrich, P8340). 50 μg of protein extract were fractionated by 8% SDS-PAGE and transferred to PVDF membrane (BioRAD, Marnes la Coquette, France). After blocking at room temperature for 2 h in TBS with 5% nonfat dry milk, the membranes were incubated for 1 h in TTBS containing 0,6 μg/ml polyclonal rabbit anti-mouse Hes1 antibody (kindly provided by T. Sudo) or polyclonal goat anti-mouse Pdx1 antibody (1:10000, kindly provided by C.V.E. Wright), and then for 1 h with TTBS containing secondary anti-rabbit (1:10000, Pierce, Perbio, Brebières, France) or anti-goat (1:10000, Dako) conjugates coupled to HRP. Antibody binding was visualized by ECL using Supersignal West Pico substrate (Pierce). Blots were stripped by 5 min incubation in 0,2 M NaOH, blocked for 1 h in TBS with 5% nonfat dry milk, and re-probed for 2 h in TTBS with primary anti-β-actin antibody (1:200, TEBU, Santa-Cruz) coupled to HRP. Anti-β-actin antibody binding was detected using ECL substrate as stated before. Band intensities were measured on a Chemidoc XRS system (BioRAD) and relative abundance of Hes1 protein normalized to β-actin was assessed by densitometry using Quantity One software (BioRAD).