Fig. 4

PTBP1 knockdown impedes autophagy flux in GC cells by upregulating TXNIP. A Reactive oxygen species (ROS) levels were detected through inverted fluorescence microscope in AGS and HGC-27 cells transfected with si-NC, si-PTBP1, or co-transfected with si-PTBP1 and si-TXNIP. Quantification of the mean gray value was presented as mean ± SD, with statistical analysis conducted using an unpaired student’s t-test, n = 3. *P < 0.05; **P < 0.01. B Lyso-Tracker Red was used to assess lysosomal abundance. Quantification of lysosome numbers was presented as mean ± SD, with statistical analysis conducted using an unpaired student’s t-test, n = 3. *P < 0.05; **P < 0.01. C Protein levels of P62, PTBP1, TXNIP, and LC3 were assessed in AGS and HGC-27 cells transfected with si-NC, si-PTBP1, or co-transfected with si-PTBP1 and si-TXNIP. β-actin served as a control. Red numbers indicated densitometric values of band intensity. D Autophagy flux was assessed in mGFP-RFP-LC3-stably expressed AGS cells transfected with si-NC, si-PTBP1, or co-transfected with si-PTBP1 and si-TXNIP. The number of LC3 puncta (yellow for autophagosomes, red for autolysosomes) was quantified. Hoechst 33342 (blue) was used for nuclear staining, and cells were imaged under a laser confocal microscope. Scale bar, 5 μm. Data were presented as mean ± SD, with statistical analysis conducted using an unpaired student’s t-test, n = 3. ***P < 0.001