Lysosensor

All cell lines were cultured at 37°C with 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone), 100 U/mL penicillin and 100 mg/mL streptomycin. No cell lines used in this study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample. All cell lines were from American type culture collection (ATCC). Transient transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturers’ instructions. TR-BSA and CA-074-Me were from Tocris Bioscience (Bristol, UK). bafilomycin A1, 3-MA, z-VAD, necrosis inhibitor IM-54 and cisplatin were from Calbiochem (Darmstadt, Germany). The mature lysosome dye BODIPY-pepstatin A, LysoSensor, Annexin V and PI were purchased from Invitrogen Life Technologies (Carlsbad, CA). The antibody against LC3 was from MBL. Antibodies against p-STAT3 and STAT3 were from Cell Signaling Technology. Mouse monoclonal antibodies for α-tubulin were purchased from Sigma-Aldrich (St. Louis, MO). HRP-, Cy3- and FITC-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).

For time-lapse microscopy, 143B cells were stained with Nile Red (1:1000, Sigma Aldrich) or Lysosensor (1:1000, Life Technologies). Frames were acquired every 60 s for 8 min. Images were acquired by confocal microscopy (Nikon, TI-E, Tokyo, Japan).

Min6 cells were cultured in 35 mm glass bottom dish or 12-well plates. Lysosome digestive function was detected using dye quenched bovine serum albumin (DQ-BSA-488 nm, Sharebio, Shanghai, China) and Lysosomal pH was detected using LysoSensor (Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated with DQ-BSA (10 µg/mL) for 6 h or LysoSensor (0.1 mg/mL) for 5 h at 37 ℃. Images were taken with confocal LSM880 (ZEISS). For lysosome digest function, fluorescence intensity at 488 nm excitation was measured. For lysosome pH, R/G ratio: the ratio of excitation 335 nm emission 452 nm (green, G) and excitation 381 nm emission 521 nm (red, R) was calculated to indicate the lysosome pH level. Fluorescent intensity quantification using Cytation5 (BioTek, Winooski, VT, USA).

To analyze the pH of endosomes containing ALIXΔPRR, HeLa MZ cells transfected with ALIXΔPRR-mCherry for 24 h were incubated for 30 min with EGF-Alexa Fluor 647 at 37°C and then chased for 90 min in marker-free medium to label late endocytic compartments. Acidic endolysosomal compartments were then labeled using 1 µM Lysosensor in Leibovitz L-15 medium (Thermo Fisher Scientific) for 5 min at room temperature. Cells were then imaged using a 100× 1.4-NA oil DIC Plan-Apochromat VC objective (Nikon) with a Nikon A1 scanning confocal microscope and Nis-elements software.