C4555

Methyl-β-cyclodextrin (MβCD) (C4555, Sigma-Aldrich) is a strictly surface-acting drug that can selectively and rapidly remove cholesterol from the plasma membrane in preference to other membrane lipids (Barman & Nayak, 2007 (link)). This cholesterol depletion reagent has been widely employed in studying the effect of both cholesterol depletion and lipid raft disassembly, and current data indicate that it inhibits entry of several viruses (Choi et al., 2005 (link); Li et al., 2007 (link); Nomura et al., 2004 (link); Imhoff et al., 2007 (link); Ren et al., 2008 (link)). To remove the plasma membrane cholesterol, concentrations of 3, 6, 9, 10, 12, 15 mM of MβCD in DMEM were prepared.
Water-soluble cholesterol (C4951, Sigma-Aldrich) was employed to replenish cholesterol after extraction of cellular and viral cholesterol using MβCD.

For investigating the entry mechanisms of RABV, we used chlorpromazine (C2481, TCI), MβCD (C4555, Sigma), nystatin (N9150, Sigma), dynasore (D7693, Sigma) and ammonium chloride (A9434, Sigma) to treat cells. N2a cell monolayers were seeded into 6-well plates or 24-well plates and pretreated with drugs as listed before for 1 h at 37 °C. After pretreatment, cells were washed with PBS and incubated with CVS at MOI of 0.1 for 1 h at 37 °C. At 3 h and 24 h postinfection (hpi), the viral RNA level was quantitated by using a reverse transcription-quantitative real-time PCR (RT-qPCR) assay and percentage of infection was observed by fluorescence microscopy. At 48 h postinfection (hpi), western blot was performed.

EVs (1 × 10⁶ per PANC-1 cell) were first labeled with PKH26 or PKH67 (Sigma-Aldrich). PANC-1 cells were seeded on the porous membrane surface of a Transwell® insert. Once the PANC-1 cells reached 90% confluence, we applied a 1% collagen solution (C5533, Sigma-Aldrich) for 1 h to seal potential leakage between the cells and the transwell before conducting the transcytosis assay. Moreover, to assess the occurrence of leakage, we compared the dtEVs that bypassed the non-cell control (only porous transwell) or PANC-1 cells with or without collagen sealing (Supplementary Fig. 30). The putative transcytosis was determined by the uptake of fluorescent-labeled EVs in cultured PANC-1 cells placed on the bottom surface of the Transwell® insert after 24 h incubation using a Spark® Cyto plate reader (Tecan, Switzerland). To determine the pathways of endocytosis or exocytosis, the PNAC-1 cells on the porous membrane surface were pre-treated with an inhibitor of Pitstop 2 (Pit2, SML1169, Sigma-Aldrich), Methyl-β-cyclodextrin (mβC, C4555, Sigma-Aldrich), Cytochalasin D (CytD, C8273, Sigma-Aldrich), Neticonazole (Ntz, SS717, Selleck), Brefeldin A (BFA, B6542, Sigma-Aldrich), or Exo1 (341220, Sigma-Aldrich) for 24 h before the transcytosis assay.

Primary and immortalized macrophages were seeded into either 6-well plates at a concentration of 2.5 × 10⁶, 12-well plates at a concentration of 1 × 10⁶, or 24-well plates at a concentration of 0.5 × 10⁶ cells/well. Where indicated, macrophages were treated with elacridar (1–10 μM, SML0486; Sigma) for 16 h. In experiments with other inhibitors, methyl-β-cyclodextrin (5–10 μM, C4555; Sigma) or MG132 (10 μM) was added 30 min before the addition of LPS. In other experiments, cells were exposed to IL-1 (2 ng/ml, 211-11B; PeproTech), cyclooxygenase 2 (COX2) inhibitor NS-398 (10 μM, 70590; Cayman), or protein kinase A (PKA) inhibitor H-89 (10 μM, 10010556; Cayman) overnight. For fatty acid supplementation, distinct fatty acids were conjugated to albumin as previously described (27 (link)). Cells were continuously cultured with albumin-bound arachidonic acid (AA; 5 μM), LA (20 μM), or a combination of AA (5 μM) and stearic acid (SA; 20 μM) for at least 2 wk before mass spectrometry (MS) analysis or any further experiments.

Different endocytotic inhibitors were tested for their optimal concentration, exposure time and cell impairment (Table 1, Figure 3). Inhibition of clathrin-mediated endocytosis in both cell types was tested with chlorpromazine hydrochloride (C8138, Sigma-Aldrich, Switzerland) and monoDansylcadaverine (Dansylcadaverine, D4008, Sigma-Aldrich). Inhibition of caveolin-mediated endocytosis was performed in both cell lines using 10 mM methyl-β-cyclodextrin (mβcd) (C4555, Sigma-Aldrich). A 4 µM cytochalasin D (C8273, Sigma-Aldrich) solution was used to inhibit phagocytosis and macropinocytosis in both cell lines.

To study the mechanisms of GBS internalization, Caco-2 cells were treated with the following chemical inhibitors: chlorpromazine (CPZ; C8138-54, Sigma-Aldrich) and monodasylcadaverine (MDC; D4008, Sigma-Aldrich) for inhibition of clathrin-mediated endocytosis; methyl-β-cyclodextrin (MβDC; C4555, Sigma-Aldrich) and genistein (Gen; G6649, Sigma-Aldrich) for inhibition of cholesterol and tyrosine kinase, respectively, in lipid rafts; cytochalasin B (CytB; C6762, Sigma-Aldrich) and nocodazole (Ndz; M1404, Sigma-Aldrich), for inhibiting cellular actin and microtubules, respectively. Cells were pre-incubated with these compounds for 1 h prior to infection at the indicated concentrations. All infections were performed in presence of inhibitors, while control cells were incubated with vehicle only. Epithelial cell viability was evaluated by trypan blue staining after incubation with the inhibitors and was always > 90%.