Anti p27kip1

Reagent and antibody sources were as follows: AG1478 (Calbiochem/Merck, Darmstadt, Germany), BMP4 (R&D Systems, Minneapolis, MN, USA), DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), temozolomide (TMZ) and anti-β-Actin-peroxidase conjugated antibody (Sigma-Aldrich, Munich, Germany), anti-AKT, anti-phospho-AKT (Ser473), anti-phospho-AKT (Thr308), anti-BIM, anti-cleaved caspase 3, anti-cleaved caspase 7, anti-cleaved PARP (poly (ADP-ribose) polymerase-1), anti-EGF Receptor, anti-phospho-EGF receptor (Tyr1068), anti-FOXO3a, anti-phospho-FOXO3a (Thr32), anti-phospho-FOXO3a (Ser253), anti-phospho-FOXO3a (Ser318/321), anti-phospho-Rb (Ser807/811), anti-SMAD1, anti-SMAD3, anti-SMAD4, anti-SMAD5, anti-phospho-SMAD1/5 (Ser463/465), anti-phospho-SMAD3 (Ser423/425), anti-p27^Kip1, anti-SOX2 (Cell Signaling Technology, Beverly MA, USA), anti-OLIG2, anti-β-Tubulin beta III isoform (Millipore, Temecula, CA, USA), anti-CYCLIN B1, p21^CIP1 (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-GFAP (BD Pharmingen San Jose, CA), anti-NESTIN (R&D Systems, Minneapolis, MN, USA), and anti-CYCLIN D1 (ThermoFisher Scientific, Waltham, MA USA).

Caco-2 and SAOs cells were incubated with CEN at the same concentrations reported above, in the presence of 2 μM ATRA, as positive control. At the end of incubation, cells were lysed using a lysis buffer containing protease and phosphatase inhibitors, as reported [53 (link)]. After the measurement of protein concentration [54 (link)], the total protein lysates were loaded on a 4%–12% precast gel (Novex Bis-Tris precast gel 4%–12%; Life Technologies) using MES (2-(Nmorpholino) ethanesulfonic acid) or MOPS [(3-(N-morpholino) propanesulfonic acid)], (50 mM MOPS, 50 mM Tris, 1% SDS, 1 mM EDTA; pH 7) buffer. The immunoblots were performed following standard procedures, using as primary antibodies: anti-LC3, anti-BECN1, anti-pAMPK^Thr172, anti p27^KIP1 (Cell Signalling Technology), and anti-α-tubulin (Sigma-Aldrich) antibodies. PVDF membranes were finally incubated with horseradish peroxidase-linked secondary antibody raised against mouse or rabbit and immunoblots developed using the ECL Plus Western blotting detection system kit (GE Healthcare, Milan, Italy). Band intensities were quantified measuring optical density on a Gel Doc 2000 Apparatus (Bio-Rad Laboratories, Milan, Italy) and multianalyst software (Bio-Rad Laboratories).

The Click-iT Plus™ EdU Alexa-Fluor-594 Imaging Kit (Life Technologies) was used to identify proliferating cells in embryonic cochlea. Pregnant females were injected with 100 µg 5-ethynyl-2′-deoxyuridine (EdU) in 200 µl PBS twice, at 2 h intervals, before embryos were harvested 2 h after final injection at either E14.5 or E18.5. Embryonic heads were fixed in 4% PFA in PBS for 1 h at 4°C and tail collected for genotyping. Fixed heads were then dehydrated and embedded in paraffin wax and 5-µm sections collected onto charged slides. The copper-azide ‘click’ Alexa-Fluor-594 reaction for detection of EdU was performed as per the manufacturer's instructions, and processed slides were washed in PBS containing 3% BSA at RT. Slides were then blocked with 1× PBS containing 5% donkey serum and 0.5% Triton X-100 (Sigma), before incubation with rabbit polyclonal anti-p27KIP1 (Cell Signaling Technology) at 1:200 dilution overnight at 4°C. Slides were washed in PBS before incubation with Alexa-Fluor-488-conjugated donkey anti-rabbit secondary antibody at 1:200 dilution. Fluorescent confocal images were collected using a Zeiss LSM 700 inverted microscope.