Fig. 2

PTBP1 Knockdown leads to an excessive accumulation of autophagosomes, blocking autophagy flux in GC cells. A Transmission electron microscopy (TEM) images showing endogenous autophagic microstructures in PTBP1-silenced and control cells. Red arrows indicated autophagosomes or autolysosomes. Scale bars, 1 or 2 μm. B AGS and HGC-27 cells were first transfected with GFP-LC3 for 24 h and subsequently transfected with either si-NC or si-PTBP1 for an additional 48 h. Formation of LC3 puncta (green) was detected using a laser confocal microscope, with Hoechst 33342 (blue) staining for nuclei. Scale bar, 5 μm. C After stable silencing of PTBP1, the colocalization of LC3B (green) and LAMP1 (red) was assessed. Hoechst 33342 (blue) was used to stain the nuclei, and images were captured using a laser confocal microscope. The white rectangle represents a partial enlargement. Scale bar, 5 μm. D Western blot was used to detect the expression levels of P62 and LC3 in PTBP1-silenced and control cells. β-actin served as a control, and densitometric values of band intensity were indicated by red numbers. E PTBP1-silenced and control cells were exposed to an mRFP-GFP-LC3 lentivirus. The number of LC3 puncta (yellow for autophagosomes, red for autolysosomes) was quantified. Hoechst 33342 (blue) was used to stain the nuclei, and images were captured using a laser confocal microscope. Scale bar, 5 μm. Data are presented as mean ± SD. Statistical analysis was performed using an unpaired student’s t-test, with n > 3. *P < 0.05; **P < 0.01; ***P < 0.001. F Western blot was used to determine the protein expression levels of P-mTOR, PTBP1, P62, and LC3. β-actin served as a control. PTBP1-silenced and control cells were treated with either DMSO or rapamycin (AGS, 50 nM; HGC-27, 250 nM) for 24 h. Data were presented as mean ± SD, with statistical analysis conducted using an unpaired student’s t-test, n > 3. *P < 0.05; **P < 0.01; ***P < 0.001