2 apb

Human ESCs were passaged with Accutase (Sigma) and plated at a density of 100,000 cells/cm² in mTeSR with 10 μM Y27632 (Selleck) on Matrigel (BD), Laminin (BD), and collagen IV (BD) (3:1:1) mixed gel coated-plate (Corning). In the restriction of definitive endoderm (DEs) stage (S1), cells were cultured for 24 hrs in RPMI with B27 supplement (1:50, Gibco), 100 ng/ml Activin A (R&D) and 3 μM CHIR99021 (Selleck), and then treated with 100 ng/ml Activin A for 2 days. In the hepatic specification stage to get hepatic progenitor cells (S2), the culture medium was replaced with RPMI (Gibco) supplemented with B27 supplement (1:50), 20 ng/ml BMP4 and 10 ng/ml FGF2 for 5 days. And in the stage of hepatic maturation (S3), cells were cultured in Hepatocyte Culture Medium (HCM, Lonza) with 20 ng/ml HGF, 10 ng/ml OSM and 1 μM dexamethasone for 10–15 days. During stages 2 and 3, cells are fed every 48 hr. The final stage was also carried out with 10 μM SB203580 (Selleck), 50 μM Vitamin K2 (Sigma), 50 μM 2-APB (Tocris), or 0.5 μM Anisomycin (Selleck), and 0.1% dimethyl sulfoxide (DSMO) was used as control, as described in the text. Cells were photographed during differentiation using a Nikon phase contrast microscope (Nikon Microscopes).

(RS)-3,5-Dihydroxyphenylglycine (DHPG), NNC 55–0396 dihydrochloride (NNC), nimodipine, 2-aminoethoxydiphenylborane (2-APB) and cycloheximide were obtained from Tocris. Rapamycin and Calyculin-A were from Cell Signaling. Calpain inhibitor III was from Calbiochem. Okadaic acid was from Santa Cruz. All the other reagents were from Sigma. Water insoluble compounds were dissolved in DMSO and diluted to reach a final concentration less than 0.1%.

A23187 (Tocris Bioscience), dantrolene (Tocris Bioscience), 2-APB (Tocris Bioscience), BAPTA-AM (Dojindo), BAPTA (Dojindo), EGTA (Dojindo), EGTA-AM (AAT Bioquest), 5,5′-difluoro-BAPTA-AM (PromoCell Gmbh), xestospongin C (Abcam), araguspongin B (Cayman Chemical), U73122 (Aobious, Gloucester), thapsigardin (Nacalai Tesque), brefeldin A (Nacalai Tesque), l-Leucyl-l-Leucine methyl ester (Cayman Chemical), antimycin A (Santa Cruz Biotechnology), and oligomycin (Calbiochem) were purchased.

Ca²⁺ imaging experiments were performed as previously described [45 (link)]. HT-29 cells cultured on coverslips were loaded with 5μM Fura-2 AM (Invitrogen, NY, USA) in physiological salt solution (PSS) at 37°C for 50 min and then washed with PSS or PPS with 2-APB (Tocris Bioscience, Minneapolis, MN, USA), a CRAC channel blockers (50μM); or GSK-7975A (Tocris Bioscience, Minneapolis, MN, USA), for 30 min. Then, cells on coverslips were placed in a standard perfusion chamber on the stage of an inverted fluorescence microscope (Nikon, Japan). For the Ca²⁺-free solution, Ca²⁺ was omitted and 0.5 mM EGTA was added to prevent possible Ca²⁺ contamination.

MSC were cultured for 21 days in αMEM with FBS (10%), ultraglutamine (1%), penicillin (100 U/mL), streptomycin (100 µg/ml) and osteogenic stimuli based on dexamethasone (1 µM; Sigma-Aldrich), β-glycerol phosphate (10 mM; Sigma-Aldrich) and ascorbic acid (0.2 mM; BAYER, Barcelona, Spain). Basal Mg²⁺ concentration in the medium was 0.8 mM. In order to increase the Mg²⁺ content in the pro-osteogenic medium, MgCl₂ (Carlo ErbaReagentiSpA, Milano, Italy) was added to achieve final Mg²⁺ concentrations of 1.2 mM and 1.8 mM during the osteogenic stimulus. Fresh medium alone or osteogenic medium supplemented with MgCl₂ was replaced every 3 days. Furthermore, 2-APB (50 µM; Tocris Bioscience, Bristol, UK) was added to the osteogenic medium containing 0.8 mM of Mg²⁺ during differentiation to determine the effects of inhibiting the Mg²⁺ channel TRPM7. To examine a direct effect of Mg²⁺ supplementation (1.2 and 1.8 m, or 2-APB) for 24 hours on undifferentiated MSC or at the different stages of differentiation (early or mature osteoblasts obtained from MSC) the NICD expression was also analyzed by western blot. All experiments were repeated at least three times.