Skip to main content
. 2018 Nov 20;14:67–79. doi: 10.1016/j.omtn.2018.11.005

Figure 7.

Figure 7

Naked and GalNAc AONs Form Cytoplasmic Vesicles of Similar Abundance, Size, and Localization in Renal Tubular Cells

(A) Abundance and size of AON-containing vesicles: PTECs were treated with Alexa 488 AONs in their naked or GalNAc-conjugated form for 60 min at 37°C prior to confocal imaging. Nuclei were revealed using NucBlue staining. Alexa 488 cytoplasmic vesicles were classified according to size (x-small: 2–3 pixels; small: 3–4 pixels; medium: 4–5 pixels; and large: ≥5 pixels). The number of vesicles of a given size was divided by the number of nuclei counted in the same field of view and reported as “number of vesicles per cell” on the y axis. Data represent mean and SD of 10 fields of view from two independent experiments. ANOVA with Sidak’s multiple comparisons test comparing GalNAc to Naked of the same AON and vesicle size group indicates no significance. (B) Co-localization of naked and GalNAc AONs: PTECs were co-treated with Alexa 488-GalNAc and Alexa Fluor 647-naked AONs for 60 min at 37°C prior to confocal imaging. Nuclei were revealed using NucBlue staining. Selected representative images show that cytoplasmic vesicles formed by GalNAc AONs (green signal, left panels) and naked AONs (red signal, middle panels) co-localize in PTECs (yellow signal, merged channel, right panels). The percentage of Alexa 488-GalNAc AON vesicles that co-localize with Alexa Fluor 647-naked AONs is reported for AON-B and AON-C. (C) Control of co-localization of AONs: Same as in (B), except that PTECs were co-treated with naked AONs only, either Alexa Fluor 647 or Alexa 488 labeled. Selected representative images reveal that Alexa Fluor 647 lead to a more diffuse and less specific signal than Alexa 488 when labeled to AONs. The percentage of Alexa 488-naked AON vesicles that co-localize with Alexa Fluor 647-naked AONs is reported for AON-B and AON-C and represent the maximum co-localization that can be detected for a given sequence and form of AON. Data represent mean and SD of 10 fields of view from two independent experiments.