Skip to main content
. 2021 May 17;17(5):e1009582. doi: 10.1371/journal.ppat.1009582

Fig 6. Exosomal circALTO can mediate ALTO expression in HeLa cells and keratinocytes.

Fig 6

(A) Western blot analysis of exosomal marker in exosomes extracted from 293 T cells transfected with pCDNA vector control or Flag-circALTO1/2. CD63 and CD81 are exosomal marker proteins, while calnexin and β-tubulin exclude contamination from the cytoplasm and cellular organelles. (B) Size distribution analysis of total exosomes isolated from the Flag-circALTO1 (upper panel) and Flag-circALTO2 (bottom panel) expressed 293T cells. (C) TEM images of exosomes isolated from circALTO2 expressed 293T cells confirm expected cup-shaped morphology. (D) RT-PCR analysis of total RNA from exosomes of transfected with pCDNA vector control or Flag-circALTO1/2 293 T cells with and without RNase R treatment. (E) Fluorescence of HeLa cells incubated with DiI labeled exosomes from indicated cells by fluorescent microscopy. Scale bar = 50 μm. (F) Purified exosomes from 293T cells transfected with Flag-circALTO1/2 were transferred to HeLa cell. Expression of Flag was assessed by WB. HSP90 is the loading control. (G) The indicated purified exosomes were transferred to HeLa cell. After 70 hours, cells were transfected with pCDNA-GFP (48 hours), then lysates were generated to determine expression of Flag and GFP by WB. HSP90 is the loading control. (H) HeLa cells treated as previously described (G) were imaged by IF. Scale bar = 200 μm. (I) The indicated purified exosomes were transferred to HeLa cell. After 70 hours, cells were transfected with pCDNA-GFP (48 hours), then lysates were generated to determine expression of Flag and GFP by WB. HSP90 is the loading control.