Journal: Scientific Reports

Article Title: Recycling endosomal CD133 functions as an inhibitor of autophagy at the pericentrosomal region

doi: 10.1038/s41598-019-39229-8

Figure Lengend Snippet: Pericentrosomal CD133 suppresses cell differentiation by inhibiting autophagy. ( A ) Representative images of primary cilium formation in RPE1 cells transfected with the CD133 (WT) or CD133 (EE) expression vector, followed by serum starvation for 24 h. CD133 is red, Arl13b (cilium) is green, and DAPI (DNA) is blue. The arrow shows a primary cilium. Arrowheads show the localisation of CD133. Scale bars, 10 μm. ( B ) Quantification of cells with primary cilia shown in ( A ); mean ± standard error of the mean (SEM) from three experiments, *p < 0.0001, *p < 0.0001, respectively. ( C ) Representative images of primary cilium formation in SK-N-DZ cells transfected with CD133 siRNA or its control, followed by serum starvation for 48 h. Pericentrin (centrosome) is green, acetylated-α-tubulin (cilium) is red, and DAPI (DNA) is blue. Arrows show primary cilia. Scale bars, 10 μm. ( D ) Quantification of cells with primary cilia shown in ( C ); mean ± standard error of the mean (SEM) from three experiments, *p = 0.0083. ( E ) Immunoblot of CD133, GAP43 (axon regeneration marker), and LC3B in SK-N-DZ cells transfected with CD133 siRNA or its control, followed by a 13-cis retinoic acid (RA) treatment for 48 h with or without chloroquine (CQ: autophagy inhibitor). An immunoblot of α-tubulin served as a loading control. ( F ) Representative images of neurite outgrowth in SK-N-DZ cells transfected with CD133 siRNA or its control, followed by a 13-cis RA treatment for 48 h with or without CQ. Scale bars, 100 μm. ( G ) Quantification of cells with neurite elongation shown in ( F ); mean ± standard error of the mean (SEM) from three experiments, *p < 0.0001, *p < 0.0001, respectively.

Article Snippet: 13-cis-retinoic acid (RA) (R0088, final 25 μM for 48 h) were obtained from Tokyo Chemical Industry.

Techniques: Cell Differentiation, Transfection, Expressing, Plasmid Preparation, Western Blot, Marker