Fig. 2. ANXA1 regulates NPC cell autophagy via a BECN1 and ATG5-dependent autophagic pathway.

a Western blot analysis showing the expression levels of BECN1, SQSTM1 and LC3-II in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. b Electron microscopic examination showing autophagic vacuoles (red arrows) in the cytoplasm of ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. Scale bar = 5 μm. c (top) Immunofluorescent staining showing the number of LC3 puncta in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. Cells was stained by indirect immunefluorescence using anti-LC3 antibody and observed by confocal microscopy (bottom). The number of LC3 puncta per cell was quantified. Scale bar = 10 μm. d ANXA1 knockdown enhances autophagic flux (left). Western blot analysis showing the LC3-II levels in the ANXA1 KD 5–8F cells and scramble shRNA control cells treated with BAF (middle and right). The number of EGFP-LC3 puncta in the ANXA KD1 5–8F cells and scramble shRNA control cells treated with BAF. Cells were transfected with 1 μg of EGFP-LC3 plasmid for 24 h, and BAF treatment for an additional 24 h, thereafter the number of EGFP-LC3 puncta was examined and quantified by fluorescence microscopy. Scale bar = 10 μm. e Autophagy induction by ANXA1 knockdwon follows an autophagic pathway dependent of BECN1 and ATG5. (left) Western blot analysis showing the LC3-II levels in the ANXA1 KD 5–8F cells and scramble shRNA control cells transfected with BECN1 or ATG5 siRNA. (middle and right) The number of EGFP-LC3 puncta in the ANXA KD 5–8F cells and scramble shRNA control cells transfected with BECN1 or ATG5 siRNA. Cells were cotransfected with 1 μg of EGFP-LC3 plasmid and siRNA against BECN1 or ATG5 for 24 h, and the number of EGFP-LC3 puncta was examined and quantified by fluorescence microscopy. Scale bar = 10 μm. Mean ± SD and statistical significance are denoted; **P < 0.01; ***P < 0.001; ns, no significance