Mouse β actin

Whole-cell lysate was taken from tumor cell cultures and cell lines when cells were 70% confluent at passage ≤5. Cells were rinsed with cold HyClone phosphate-buffered saline (PBS; Fisher Scientific, Waltham, MA, USA), scraped, and lysed with radioimmunoprecipitation (RIPA) buffer supplemented with a cocktail of protease inhibitors and serine/threonine and tyrosine phosphatase inhibitors (Fisher Scientific). Protein supernatants were separated by 7.5% Mini-Protean pre-cast gels (Bio-Rad, Hercules, CA, USA) at 120 V for 1.5 h then transferred onto a PVDF membrane at 100 V for 1 h. The membrane was then blocked with 5% nonfat skim milk in TBS-T for 1 h then incubated overnight in primary antibody. Primary antibodies used were: mouse β-actin (1:10,000, A1978, Sigma Aldrich, St Louis, MO, USA); mouse IKKβ (1:250, IMG-129A, Novus Biologicals, Littleton, CO, USA); mouse myosin heavy chain (clone MF20) (1:500, MAb4470, R&D Systems, Minneapolis, MN, USA); and phospho-p65 (1:1000, 4025, Cell Signaling, Danvers, MA, USA).

mRNA expression of Hdac7 and Tcf7l2 was analysed using TaqMan assays and related to expression of Ppia (Life Technologies) by quantitative real-time (q)PCR and the ΔΔC_t method. To verify overexpression of HDAC7 protein, clonal beta cells were transfected with haemagglutinin-tagged cDNA for Hdac7 and lysed in RIPA buffer (50 mmol/l Tris, pH 7.6, 150 mmol/l NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-X100, protease inhibitor cocktail; Sigma-Aldrich, St Louis, MO, USA), and boiled with sample buffer (60 mmol/l Tris, pH 6.8, 10% glycerol, 2% SDS, 10% β-mercaptoethanol, bromophenol blue). Samples were separated on Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA) and transferred onto Hybond-LFP PVDF membranes (GE Healthcare, Piscataway, NJ, USA). Protein expression was detected using a rabbit haemagglutinin tag (Abcam, Cambridge, UK; diluted 1:4000) and mouse β-actin (Sigma-Aldrich; diluted 1:10,000) antibodies, and secondary DyLight 680/800 conjugated goat antibodies (Thermo Scientific, Rockford, IL, USA; diluted 1:15,000), all validated by the respective suppliers. Blots were scanned using an Odyssey imaging system (LI-COR, Lincoln, NE, USA).

All cell culture media and supplements were purchased from Biochrom AG. Unless otherwise stated, all other reagents were from Sigma–Aldrich. Primary rabbit anti-TRPV6 antibody was purchased from Santa Cruz Biotechnology. Mouse β-actin and all secondary antibodies were purchased from Sigma–Aldrich.

All reagents were from Sigma unless otherwise stated. Standard protocols were used as previously described42 (link)59 (link). Mouse β-actin, 1:10000, Sigma, A5441; Mouse HIF-1α,1:1000, BD Pharmingen, 610958; Rabbit HIF-2α, 1:1000, Novus Biologicals, NB100-122; Rabbit REST, 1:1000, Abcam, ab28018.