Journal: EMBO Molecular Medicine
Article Title: Extracellular vesicle-based targeted protein degradation platform for multiple extracellular proteins
doi: 10.1038/s44321-025-00371-8
Figure Lengend Snippet: ( A ) The uptake levels of specific antibody-blocking EVs (IgG, CD71, SQSTM1, TMEM59, CCN1, ITM2B, ITM2C) were detected after incubation for 12 h. IgG control was used to normalize the grayscale values of each group. Data were analyzed by one-way ANOVA ( n = 3). ( B ) The uptake levels of specific gene-knockdown EVs (Vector, CD71, SQSTM1, TMEM59, CCN1, ITM2B, ITM2C) were detected after incubation for 12 h. Vector control was used to normalize the grayscale values of each group. Data were analyzed by one-way ANOVA ( n = 3). ( C ) Control EVs and SQSTM1 KD EVs were incubated with NPCs for 12 h. Uptake level was analyzed at this time while degradation level was detected following wash out and another 12 h incubation ( n = 3). ( D ) IEM assay to detect the expression of SQSTM1 on HEK-293T derived EVs (scale bar: 100 nm). ( E ) Anti-SQSTM1 antibody was used to pull down HEK-293T derived EVs. IgG was used as negative control. SQSTM1 as well as EV marker-CD63 were detected by western blot. ( F ) Control or SQSTM1 KD EVs were added to NPCs and co-incubated for 12 h. Then, free EVs were wash out and replaced by free mediun containing 50 nM BafA1 for 12 h. After incubation, lysosome enrichment method was used to detect the sorting efficiency of control and SQSTM1 knockdown EVs into lysosomes in NPCs ( n = 3). ( G ) Control or SQSTM1 KD EVs were added to NPCs and co-incubated for 12 h. Then, free EVs were washed out and replaced by free mediun containing 50 nM BafA1 for 12 h. After incubation, immunofluorescence assay was used to detect the co-localization of EGFP with LampII (scale bar: 10 µm) ( n = 6); ( H ) Control or SQSTM1 KD EVs were added to HeLa cells and co-incubated for 12 h. Then, free EVs were washed out and replaced by free mediun containing 50 nM BafA1 for 12 h. After incubation, lysosome enrichment method was used to detect the sorting efficiency of control and SQSTM1 knockdown EVs into lysosomes in HeLa cells ( n = 3); ( I , J ) Analysis of degradation rate ( I ) and lysosomal sorting efficiency ( J ) of control and SQSTM1 overexpression EVs in NPCs ( n = 3); ( K , L ) Analysis of degradation rate ( K ) and lysosomal sorting efficiency (l) of control and SQSTM1 overexpression EVs in HeLa cells ( n = 3). Data were analyzed by one-way ANOVA ( A , B ) unpaired two-tailed t tests ( C , F , G , H , I , J , K , L ). Data were shown as mean ± SD. Each n in ( A , B , C , F , G , H , I , J , K , L ) is biological independent samples. The P values are labeled in the figure.
Article Snippet: CD71 Polyclonal antibody , Proteintech , 10084-2-AP.
Techniques: Blocking Assay, Incubation, Control, Knockdown, Plasmid Preparation, Expressing, Derivative Assay, Negative Control, Marker, Western Blot, Immunofluorescence, Over Expression, Two Tailed Test, Labeling
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