Lamp1

Ovarian cancer cells were harvested and lysed with RIPA buffer, consisting of 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium orthovanadate (Na₃VO₄), and 1X protease inhibitor cocktail. The lysates were centrifuged at 12,000× g, 4 °C for 15 min, and the supernatant (whole protein) was transferred to new tubes. Protein quantification was performed using a Pierce BCA protein assay kit (Thermo Fisher Scientific). A total 10 µg of protein per well was separated using 6–10% SDS-PAGE and then transferred to 0.45 µm poly-vinylidene fluoride (PVDF) membranes (Bio-Rad). After blocking with 5% skim milk solution, the membrane was probed with primary antibodies, including TRPML1 (Sigma-Aldrich), LAMP1 (Santa Cruz Biotechnology, Dallas, TX, USA), MDR1 and Actin (Cell Signaling Technology, Danvers, MA, USA). Following incubation with secondary antibodies conjugated with horseradish peroxidase (HRP), signals were visualized using a chemiluminescence detection kit (Westar ƞC ultra 2.0, Cyanagen, Bologna, Italy). Images were captured by a chemiluminescence imaging system (Vilber, The FUSION Solo X, Eberhardzell, Germany).

For mitochondrial staining, cells were stained with the mitochondrial probe, MitoTracker Red CMXRos (Thermo Fisher Scientific), according to the manufacturer’s protocol. The stained cells were washed twice with PBS and fixed in 4% paraformaldehyde for 15 min. The fixed cells were washed twice with PBS, blocked in 1% BSA solution, and incubated overnight with LC3B (Cell signaling Technology) and LAMP1 (Santa Cruz Biotechnology, TX, USA) at 4 °C. The cells were then washed twice with PBS, incubated with FITC-conjugated secondary antibodies (chicken anti-mouse and goat Alexa Fluor 488; Invitrogen, MA, USA) for 2 h, and again washed twice with PBS. Thereafter, the cells were washed with PBS and Fluorescent Mounting Medium with DAPI (GBI Labs, WA, USA), and then imaged by confocal microscopy (Carl Zeiss, Oberkochen, Germany) with Zeiss microscope image software ZEN (Carl Zeiss). The co-localization coefficient levels were quantified with Image J (NIH).

Samples were run on 4–12% bis-tris polyacrylamide gels (ThermoFisher), transferred to Immobilon-FL PVDF (MilliporeSigma), and probed overnight with primary antibody. The following primary antibodies were used: rabbit polyclonal GCase (#G4171, MilliporeSigma), mouse monoclonal GCase (#sc-166,407, Santa Cruz Biotechnology), cathepsin D (goat polyclonal, #sc-6486, Santa Cruz Biotechnology), LAMP-1 (mouse monoclonal, #sc-20,011, Santa Cruz Biotechnology), LAMP-2 (mouse monoclonal, #sc-18,822, Santa Cruz Biotechnology), GAPDH (mouse monoclonal, #MAB374, MilliporeSigma), Flag (mouse monoclonal, #F3165, MilliporeSigma), and progranulin (rabbit polyclonal, #40–3400, ThermoFisher). Blots were then probed with species-matched IRdye-conjugated secondary antibodies (Li-COR Biosciences) and scanned on an Odyssey scanner (Li-COR Biosciences).