Journal: Nucleic Acids Research

Article Title: Golgi-endosome transport mediated by M6PR facilitates release of antisense oligonucleotides from endosomes

doi: 10.1093/nar/gkz1171

Figure Lengend Snippet: GCC2 localizes to LEs in HeLa and SGVA cells treated with PS-ASO. ( A ) Immunofluorescent staining of GCC2 in HeLa cells. Scale bar, 10 μm. ( B ) Immunofluorescent staining of GCC2 in HeLa cells incubated with 2 μM Cy3-labeled PS-ASO 446654 for 18 h. Scale bars, 10 μm. The regions boxed with dashed lines are magnified in the insets. ( C ) Immunofluorescent staining of GCC2 and LAMP1 in HeLa cells. Scale bars, 10 μm. ( D ) Immunofluorescent staining of GCC2 and LAMP1 in HeLa cells incubated with Cy3-labeled PS-ASO 446654 for 18 h. Scale bars, 10 μm. ( E ) 3D image of GCC2, PS-ASO and LAMP1 co-localization in HeLa cells incubated with Cy3-labeled PS-ASO 446654 for 18 h. The image was compiled from 25 sections taken at 0.1 μm depth. Two vesicles are marked with red and white arrows. ( F ) Immunofluorescent staining of GCC2 in GFP-Rab7-expressing SVGA cells incubated with 2 μM Cy3-labeled PS-ASO 446654 for 18 h. Scale bars, 5 μm. The regions boxed with dashed lines are magnified in the insets. The co-localization of GCC2 with PS-ASOs inside an LE is marked with an arrow.

Article Snippet: For live cell imaging, GFP-Rab7-expressing SVGA cells grown in glass-bottom dishes were incubated with 2 μM PS-ASO 446654 for 6 h. For GFP-M6PR-CD expression, plasmid (RG201277, Origene) was transfected into HeLa cells for 24 h. Cells were reseeded into glass-bottom dish and incubated for ON.

Techniques: Staining, Incubation, Labeling, Expressing

Journal: Nucleic Acids Research

Article Title: Golgi-endosome transport mediated by M6PR facilitates release of antisense oligonucleotides from endosomes

doi: 10.1093/nar/gkz1171

Figure Lengend Snippet: M6PR co-localizes with PS-ASOs and GCC2 in LEs in SVGA and HeLa cells. ( A ) Immunofluorescent staining of M6PR-CI in GFP-Rab7-expressing SVGA cells. Scale bars, 10 μm. In the far right panel, the region boxed with a dashed line is magnified in the inset. Co-localization between Rab7 and M6PR-CI is marked by arrows. ( B ) Immunofluorescent staining of M6PR-CI in GFP-Rab7-expressing SVGA cells incubated with 2 μM Cy3-labeled PS-ASO 446654 for 6 h. Scale bars, 10 μm. The regions boxed with dashed lines are shown at higher magnification in insets, and co-localization is indicated by arrows. ( C ) Quantification of co-localization between Rab7 and M6PR-CI in cells incubated with or without ASO for 6 h. Error bars are standard deviations. P -value was calculated based on unpaired t -test. ****, P < 0.0001. ( D ) Immunofluorescent staining of M6PR-CI and GCC2 in HeLa cells incubated with 2 μM Cy3-labeled PS-ASO 446654 for 12 h. An example of co-localization in a cytoplasmic area is boxed. ( E ) 3D image of the boxed area in panel D. Two foci where M6PR-CI, GCC2, and PS-ASO co-localize are marked by arrows.

Article Snippet: For live cell imaging, GFP-Rab7-expressing SVGA cells grown in glass-bottom dishes were incubated with 2 μM PS-ASO 446654 for 6 h. For GFP-M6PR-CD expression, plasmid (RG201277, Origene) was transfected into HeLa cells for 24 h. Cells were reseeded into glass-bottom dish and incubated for ON.

Techniques: Staining, Expressing, Incubation, Labeling

Journal: Nucleic Acids Research

Article Title: Golgi-endosome transport mediated by M6PR facilitates release of antisense oligonucleotides from endosomes

doi: 10.1093/nar/gkz1171

Figure Lengend Snippet: M6PR-CI co-localizes with PS-ASOs inside LEs and on the membranes of LEs. ( A ) Immunofluorescent staining of M6PR-CI in GFP-Rab7-expressing SVGA cells incubated with 2 μM PS-ASO 446654 for 12 h. Scale bars, 10 μm. The regions boxed with dashed lines are shown at higher magnification in insets. M6PR-CI co-localization with a distinct PS-ASO-containing focus inside the LE is marked with an arrow. ( B ) Immunofluorescent staining of M6PR-CI in GFP-Rab7-expressing SVGA cells incubated with 2 μM PS-ASO 446654 for 12 h. Co-localization of M6PR and PS-ASOs as a distinct structure on the LE membrane is marked by arrows. Scale bars, 5 μm. ( C ) Live cell imaging of a SGVA cell incubated with PS-ASO 446654 for 6 h. The nuclear border is marked by a dashed line. The boxed PS-ASO-containing LE in the lower right is magnified in panel D. ( D ) Upper images: Snapshots taken during live cell imaging. Potential PS-ASO release events are marked by arrows. Lower plots: Signal intensity profiles for the LE across the lines as indicated in the upper panels. Arrows indicate ASOs outside the LE body.

Article Snippet: For live cell imaging, GFP-Rab7-expressing SVGA cells grown in glass-bottom dishes were incubated with 2 μM PS-ASO 446654 for 6 h. For GFP-M6PR-CD expression, plasmid (RG201277, Origene) was transfected into HeLa cells for 24 h. Cells were reseeded into glass-bottom dish and incubated for ON.

Techniques: Staining, Expressing, Incubation, Live Cell Imaging