Methyl β cyclodextrin

Cells were suspended in DMEM or IMDM at 1.0 × 10⁶ cells/mL in 1.5 mL microtubes. After the uptake of talaporfin, the following endocytosis inhibitors were added: 2‐deoxyglucose (2‐DG; Sigma‐Aldrich, St Louis, MO) at a concentration of 50 mmol/L), sodium azide (0.1% w/v; Kanto Chemical, Tokyo, Japan), methyl‐β‐cyclodextrin (5 mmol/L; Wako, Osaka, Japan), genistein (0.1 mmol/L; Wako), sucrose (0.45 M; Wako), chlorpromazine hydrochloride (20 mmol/L; Tokyo Chemical, Tokyo, Japan), cytochalasin β (1 mmol/L; Wako) and colchicine (1 mmol/L; Wako). After culture at 37°C for 30 minutes, the cells were chilled on ice for 30 minutes, washed three times with PBS, fixed with 0.5% formalin and analyzed by flow cytometry. For the inhibition of clathrin‐dependent endocytosis, Pitstop 2 (ab120687; Abcam, Cambridge, UK) was added to the serum‐free DMEM or IMDM to a final concentration of 25 μmol/L, and the cells were cultured at 37°C for 1 hour. Talaporfin was subsequently added to the cells to a final concentration of 30 μg/mL and the cells were washed three times with PBS, fixed with 0.5% formalin, and analyzed by flow cytometry.

The PLA was performed using a Duolink PLA kit (Sigma-Aldrich) according to the manufacturer's instructions and as previously described (Arasaki et al., 2015 (link)) except for the use of anti–MT1-MMP (R&D Systems) and anti-GFP (Thermo Fisher Scientific), or anti-mCherry (GeneTex) and anti-FLAG M2 (Sigma-Aldrich). Cholesterol depletion was performed as described previously (Ohtani et al., 1989 (link); Rothberg et al., 1992 (link)). Briefly, cells were spread on fibronectin-coated coverslips and left for 7 h, and then treated with 5 μM methyl-β-cyclodextrin (Wako Pure Chemicals) or 50 μg/ml nystatin (Sigma-Aldrich) for 30 min at 37°C. After that, the cells were fixed and subjected to PLA reactions. PLA signals were imaged by confocal microscopy and quantified by using ImageJ software.

The following reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan): 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), acetonitrile, methanol, methyl tert-butyl ether (MTBE), methyl-β-cyclodextrin, β-carotene, trifluoroacetic acid (TFA), and pyrogallol. Fluorescein and Folin-Ciocalteu reagent were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Additionally, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), and β-cryptoxanthin was purchased from Funakoshi Co., Ltd. (Tokyo, Japan).