Antifade mounting medium with hoechst 33342

U251 cells stably expressing the mCherry‐GFP‐LC3 reporter construct were subjected to lentivirus transduction to knockdown IGF2BP2 using shRNA. In the mCherry‐GFP‐LC3 reporter assay, the green fluorescent spots of GFP, which are sensitive to the acidic environment of lysosomes, indicate the presence of autophagosomes, while the red fluorescent spots of mCherry, being more stable, signify autolysosomes. Following the knockdown of IGF2BP2, cells were fixed using 4% paraformaldehyde and mounted using Antifade Mounting Medium with Hoechst 33342 (Cat# C1025; Beyotime) for nuclear staining. Subsequently, confocal microscopy (Olympus IXplore SpinSR Super Resolution Microscope) was employed to capture fluorescent images of the stained cells. The fluorescent spots were then quantified and analyzed to assess the generation of autophagosomes and autolysosomes.

Cells were seeded onto 6-well plates containing coverslips. When the cell confluency reached approximately 80%, the cells were fixed with 4% paraformaldehyde. Subsequently, the cells were washed with PBS and permeabilized using 0.1% Triton X-100. After another wash with PBS, the cells were blocked with 1% BSA to prevent nonspecific binding. Next, the cells were incubated with the primary antibody at 4°C overnight. Following the overnight incubation, the cells were washed again with PBS to remove any unbound primary antibody. Subsequently, the cells were incubated with the secondary antibody labeled with Alexa Fluor 488 (Absin, Cat# abs20013, China) or 594 (Absin, Cat# abs20021, China) in the dark at room temperature for 1 hour. The coverslips were mounted using Antifade Mounting Medium with Hoechst 33342 (Beyotime, Cat# C1025, China). Confocal microscopy was performed to capture fluorescent images of the stained cells.

Fluorescein-labelled HBP1 was incubated with different polymers and added into cells 8 h, 4 h, 2 h, and 1 h before final treatment. Then, treated cells were washed with cold PBS twice and stained with lysotracker Red (Beyotime, Shanghai) for 1 h. Next, we used Antifade Mounting Medium with Hoechst 33342 (Beyotime, Shanghai) to stain the nuclei and observed by confocal laser scanning microscopy (TCS, SP8, Leica, Germany).