Amiloride

PMs were plated in 6-well plates or 24-well plates and pretreated with different inhibitors for 30 min at 37°C, including amiloride (1.5 mM, Selleckchem, USA), chlorpromazine hydrochloride (CPZ, 15 μg/mL, Selleckchem, USA), genistein (150 μM, Selleckchem, USA), and methyl-β-cyclodextrin (M-β-CD, 12mM, Sigma-Aldrich, Merck, Germany). amiloride, chlorpromazine hydrochloride (CPZ), genistein, and methyl-β-cyclodextrin (M-β-CD) were the inhibitors of micropinocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, and lipid raft-mediated endocytosis, respectively. Subsequently, PMs were transfected with CLP/siRNA (10:1, w/w) for 48 hours. The transfection efficiency was simultaneously detected by FCM and immunofluorescence (IF). For FCM, PMs were digested with Trypsin-EDTA solution (Servicebio, China) at 37°C for 10 min, and then cells were collected to detect the fluorescence intensity of FAM bound to siNLRP3. In addition, PMs cell membranes were stained with Dil (10 mg/mL, Beyotime, China), and nuclei were stained with Hoechst (1 mg/mL, Solarbio LIFE SCIENCES, China). Fluorescence intensity was detected by confocal microscopy (NIKON-A1R/STORM, NIKON, Japan).