8 well chambered coverglass

A total of 50,000 cells were seeded in complete RPMI medium in 8-well chambered coverglass (ThermoFisher Scientific, Madrid, Spain) and incubated overnight at 37 °C and 5% CO₂ to allow cell adhesion. Cells were incubated with 5-DTAF-fluorescently labelled PM (10 mg/mL) for 6 h. Lysosomes were stained with 1 µM Lysotracker^® Red DND-99 (ThermoFisher Scientific Madrid, Spain) for 30 min at 37 °C while the cell membrane was stained with 5 µg/mL CellMask™ (Invitrogen, ThermoFisher Scientific, Madrid, Spain) for 15 min at 37 °C. Subsequently, cells were fixed in 4% PFA (Merck Life Science S.L.U., Madrid, Spain) at 4 °C for 20 min followed by nuclei staining with DAPI (1 μg/mL) for 5 min at RT in the dark. Cells were viewed under a Spectral Confocal Microscope MFV1000 Olympus (Olympus Iberia, S.A.U., L’Hospitalet de Llobregat, Spain). The 561 nm excitation wavelength of the green laser (10 mW) was used for selective detection of the red fluorochromes (Lysotracker^® Red and CellMask™). The 488 nm excitation wavelength of Argon multiline laser (40 mW) was used for selective detection of the green fluorochrome (5-DTAF). The nuclear staining DAPI was excited at 405 nm with a violet laser (6 mW). Minimal single optical sections were collected for each fluorochrome sequentially. Images were merged and analyzed with Image J 1.49v software.

(i) Trypsinized HEK293T cells (4 × 10⁵) were incubated with CV-labeled BNCs in PBS (100 µL) in a microtube on ice for 1 h and they were washed twice with cold PBS. Cells were suspended in a small volume of PBS and then an aliquot of the suspension on a cover glass was immediately subjected to LSM (FV-1000, Olympus). (ii) To examine colocalization of CV-labeled BNCs and SR-B1-GFP in BNC binding, HEK293T, SR-B1-HEK, and GFP-HEK cells were precultured for 24 h in the RPMI1640 medium (1 × 10⁵/mL, 300 µL/well) in an 8-well chambered cover glass (Thermo Fisher Scientific) that was coated with poly-L-lysine. After cells were gently washed twice with cold PBS, they were incubated with CV-labeled BNCs at 4 °C for 30 min. Cells were washed twice with cold PBS and they were subjected to LSM immediately.

Mammary tumors were digested with the Mouse Tumor Dissociation Kit (130-096-730, Miltenyi) in a gentleMACS Dissociator (130-095-937, Miltenyi) following manufacturer’s instructions. Cells were cultured on 8-well chambered coverglass (Thermo Scientific, 155411) in DMEM supplemented with 10% tet-Free Serum, 4mM L-glutamine (GIBCO), 100 μg/ml Penicillin/Streptomycin (GIBCO), 5 µg/ml insulin (Sigma), 10 µg/ml EGF (Sigma), 1 µg/ml hydrocortisone (Sigma), 1 µM progesterone (Sigma), 5 µg/ml prolactin (NIHPP), and 1 μg/ml doxycycline. Time-lapse imaging during 15 h was performed on a Zeiss Cell Observer with 2 μm optical sectioning across 18 μm stack, 30 frames/h. Zeiss Zen 2 software served for image analysis.

Murine neuroblastoma Neuro-2a (N2a) cells were obtained from American Type Culture Collection (ATCC; Manassas, VA) and maintained in DMEM (D5796, Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (12676029, Thermo Fisher Scientific, Waltham, MA), 100 U/mL penicillin G (Sigma-Aldrich) and 0.1 mg/ml streptomycin (Sigma-Aldrich) as previously described^{34 (link)}. One day before transfection 2.0 × 10⁵ N2a cells were transferred to a glass bottom dish (3910–035, IWAKI-AGC Technoglass, Shizuoka, Japan) for confocal imaging and FCS experiments or an 8-well chambered coverglass (155411, Thermo Fisher Scientific) for FLIM experiments. Plasmid DNAs were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. After overnight incubation, the transfection medium was replaced with Opti-MEM I Reduced serum medium (Opti-MEM; 31985070, Thermo Fisher Scientific) for control experiments, which was thereafter replaced with Opti-MEM containing 290 mM NaCl for the hyperosmotic stress experiments.

Cells were grown on the 8-well chambered coverglass (Thermo Fisher Scientific, Waltham, MA, USA) for 36 h. Immediately after adding the compound with indicated concentrations to cells, a small region of interest (ROI) was randomly chosen and irradiated using the laser line at 552 nm from a confocal microscopy (Leica TCS SP8 STED, Germany) over 10 min. During that period, the fluorescent and differential interference contrast (DIC) images were acquired at 0 min, 5 min and 10 min, respectively. The power of the laser was kept at 80% output to ensure the consistent irradiation between experiments.

Cells were seeded on an 8 well-chambered coverglass (155411, Thermo Fisher Scientific) and imaged on a Carl Zeiss LSM 880 confocal microscope using a 40 × 1.4 NA oil immersion objective. Before the imaging was performed, cell culture media was replaced with phenol red-free media. 50 nM LysoTracker™ Deep Red (L12492, Invitrogen) was directly added to the cell culture media and incubated for 15–60 min before the image acquirement. For RAB5/RAB7 co-localization experiment, RAB5-RFP or RAB7-RFP plasmids (14437 and 14436, Addgene) were transfected to PSAP-OE cells 24 h before the experiment using Lipofectamine 2000 (11668019, Invitrogen) as per manufacturer instructions.