Methyl β cyclodextrin mcd

SH-SY5Y cells were seeded on 24-well plates and transfected in triplicates with 200 ng of wt Wnt3-GFP or Wnt3S212A-GFP or 75 ng of membrane-bound GFP as control together with 20 ng of firefly luciferase reporter pGL3 BAR (Biechele and Moon, 2008 (link)) and 5 ng of renilla luciferase reporter pGL4.73 hRLuc/SV40 (Promega, Madison, WI, USA) using Fugene HD Transfection Reagent (1 μg/1 μL, Promega). Twenty-four hours after transfection, reporter activity was measured using the dual luciferase reporter assay kit (Promega, Madison, WI, USA). Statistical analysis was performed using Student's t-test. Error bars represent SD, where ^***p < 0.001, ^**p < 0.01, and ^*p < 0.05. HEK293T cells were transfected with wt Wnt3-EGFP or S212A Wnt3-EGFP by electroporation using the Neon® Transfection System (Invitrogen, Singapore) according to the manufacturer's protocol. The transfected cells were incubated for ~24 h, washed with Hank's Balanced Salt Solution (HBSS; Invitrogen, Singapore) twice, and imaged using imaging medium (Phenol red free DMEM + 10% FBS). For cholesterol extraction measurements, 3 mM methyl-β-cyclodextrin (MßCD; Sigma-Aldrich) dissolved in HBSS was added to cells and incubated for 15 min. The treated cells were then washed with HBSS twice and measured in imaging medium.

Laurdan, M-laurdan and C-laurdan were all synthesised in-house as previously described (
Mazeres
et al., 2014). Aliquots of 2 mM stock solutions in DMSO were kept in long-term storage at -20°C; when needed for experiments, aliquots were kept at 4°C for, at most, six weeks. Various staining procedures were used for different experiments, as described later. Nile Red was obtained from Sigma; staining was performed at 10ng/ml for 30–60 min at 37°C in tissue culture medium without serum. LysoTracker Red DND-99 was obtained from Invitrogen; staining was performed at 20 ng/ml (50 nM) for 30–60 min at 37°C in tissue culture medium without serum. Methyl-β-cyclodextrin (MßCD) (cell culture tested) was obtained from Sigma.

Radiolabeled [³H]-Taurocholic acid was purchased from PerkinElmer (Boston, MA, USA). Radiolabeled [³H]-Methyl-4-phenylpyridinium iodide (MPP⁺) was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA). Methyl-β-cyclodextrin (MCD) and water-soluble Cholesterol-methyl-β-cyclodextrin (cholesterol) were acquired from Sigma Aldrich (St. Louis, MO, USA). Taurocholic acid sodium salt (97% pure) was purchased from Sigma Aldrich.
Rabbit anti-Flotillin-1 antibody (F1180, 1:2000) was obtained from Sigma Aldrich. Mouse anti-Clathrin Heavy Chain antibody (610499, 1:1000) and mouse anti-Caveolin 1 antibody (610406, 1:1000) were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Mouse anti-Alpha 1 Sodium Potassium ATPase antibody (ab7671, 1:2000) was purchased from Abcam (Waltham, MA, USA). Mouse Tetra His antibody (34670, 1:2000) was bought from QIAGEN (Germantown, MD, USA). The mouse anti-SLC22A1 antibody was obtained from Novus Biologicals (Littleton, CO, USA).