Rhodamine 110

POPC, DOPC, 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’,5’-bisphosphate) (di18:1 PI(4,5)P₂), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’,5’-bisphosphate) (18:0 20:4 PI(4,5)P₂), 1-oleoyl-2-{6-[4-(dipyrrometheneboron difluoride)butanoyl]amino}hexanoyl-sn-glycero-3-phosphoinositol-4,5-bisphosphate (TopFluor PI(4,5)P₂), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (biotinylated DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 1,2-Dipalmitoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’,5’-bisphosphate) (di16:0 PI(4,5)P2) was from Echelon Biosciences (Salt Lake City, UT, USA). Lipid stock solutions were prepared in chloroform, with the exception of the phosphoinositides, which were prepared in chloroform:methanol (MeOH) 2:1 (v/v). Both solvents were obtained from Merck (Darmstadt, Germany) and were of spectroscopic grade. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ethanol (EtOH), NaCl, Sucrose, EDTA, glucose, and CaCl₂ were from Sigma-Aldrich (St. Louis, MO, USA). TMA-DPH, tPnA, Rhodamine 110, and Fluo-5N were from Molecular Probes, Invitrogen (Eugene, OR, USA).

The pancreatic acinar cells were grouped and treated as follows: Control: no treatment. Caerulein: pancreatic acinar cells were induced with caerulein (1 × 10⁻⁷M) for 5 h (Zhang et al., 2022 (link)). Caerulein + miRNA-NC group: pancreatic acinar cells were first treated with miRNA-NC transfection, and then stimulated with caerulein. Caerulein + miR-455-3p mimics group: pancreatic acinar cells were first treated with miR-455-3p mimics transfection, and then stimulated with caerulein. After the cells were digested by trypsin, the liquid was centrifuged, and the cells precipitates were collected. Then, the pancreatic acinar cells were resuspended with HEPES buffer containing 2mM EDTA (HBS, 5mM HEPES, 0.15M NaCl, PH 7.35). The cell density was controlled at 1 × 10⁶ cells/mL (Zhang et al., 2019 (link)), and 10 µM Rhodamine 110, bis-(p-tosyl-L-glycyl-L-prolyl-L-arginine amide) (R22124; Molecular Probes, Eugene, OR, USA) was added for 20 min of incubation. The fluorescence intensity was detected by laser confocal microscopy and the activity of trypsin was analyzed.

1-Palmitoyl-2-oleoyl-sn-glicero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5′-bisphosphate) (PI(4,5)P₂), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (biotinylated DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Lipid stock solutions were prepared in chloroform with the exception of the PIs that were prepared in chloroform and methanol (MeOH) (2:1 vol/vol). Both solvents were obtained from Merck (Darmstadt, Germany) and were of spectroscopic grade. Phosphate-buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ethanol (EtOH), NaCl, sucrose, glucose, CaCl₂, imidazole, glycerol, bovine serum albumin (BSA), BSA-biotin, and avidin were purchased from Sigma-Aldrich. Rhodamine 110 and Fluo-5N were obtained from Molecular Probes, Invitrogen (Eugene, OR, USA).