Dq bsa

Phagosomal proteolytic activity was measured by feeding cells DQgreen/Alexa Fluor 594 (DQ-BSA; Invitrogen, D12050) co-labelled 3-μm silica beads (Kisker Biotech, PSI-3.0COOH) as previously described [76]. Briefly 3 × 10⁵ cells/well were seeded in a 96-well plate before addition of beads, and fluorescence measured on a plate reader each minute in triplicate. Proteolysis was normalised to Alexa Fluor 594 fluorescence, over time to account for potential differences in bead uptake and rates normalized to wild-type cells to calculate relative activity.

WT 3T3 cells (5 × 10⁴ cells) were grown on coverslips and treated with MVO 50 μM for 16 h. Then, cells were incubated with 10 μg/ml of DQ-BSA (Invitrogen) or 50 μg/ml of FiTC-BSA for 2 h at 37 °C, washed twice with PBS, then fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, mounted using Vectashield mounting medium with DAPI (Vector Laboratories) and images were obtained using an LSM 700 confocal microscope (Carl Zeiss).

The following primary antibodies were used in immunofluorescent staining and western blotting: anti-Lamp-1 (1D4B, H4A3, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti-Cathepsin D (C-20, sc6486, Santa Cruz Biotechnology, Dallas, TX, USA) anti-Cathepsin B (T-12, sc86313) and anti-GAPDH (H-12, sc166574), anti-TFEB (Proteintech, 13372-1-AP, Rosemont, IL, USA). HRP conjugated secondary antibodies were purchased from Santa Cruz.
Texas Red 10 kD Dextran (Invitrogen, 1 mg/mL) was used to label lysosomes. LysoTracker Red DND-99 (Invitrogen, 50 nM) was used to indicate acidic lysosomes. DQ-BSA (Invitrogen, 10 μg/mL) was used to detect lysosomal function. BODIPY FL-pepstatin A (Invitrogen, 1 μM) was used to stain active Cathepsin D. A Magic Red™ Cathepsin B Assay Kit (ImmunoChemistry Technologies, Bloomington, MN, USA) was used to stain active Cathepsin B. 4, 4′-Diisothiocyano-2, 2′-stilbenedisulfonic acid (DIDS, 5 μM, Tocris Bioscience, Bristol, UK) was used to inhibit SLC17A9.

To measure lysosomal protease activity, DQ-BSA (Invitrogen #D12050) was dissolved at 1 mg/mL in DPBS by water-bath sonication. Cells were plated in glass-bottom 12-well plates and grown to 60–70% confluency. Cells were washed with DPBS and fed with DQ-BSA (Invitrogen #D12050) at 100 μg/mL in complete DMEM medium for 4 h and treated with LysoTracker Red DND-99 (Invitrogen #L7528) for 15 min and subsequently imaged on a Nikon CSU-W1 SoRa microscope. Cells were maintained in a live cell chamber at 37°C, 85% humidity and 5% CO₂.

To assess endocytic cargo degradation, 40,000-50,000 WT or DENND6A KO cells were seeded on PLL-coated coverslips within a 4-chambered dish. Subsequently, the cells were incubated in culture media containing 20 µg/mL of DQ-BSA (D12051, Invitrogen) for 2 hours at 37 °C. After the incubation, the culture media was removed, and the cells were fixed with 4% paraformaldehyde at room temperature, followed by DAPI staining. Finally, coverslips were mounted onto microscopic slides using fluorescence mounting medium (Dako, catalog no. S3023).