Activated stat5 trafficking via endothelial cell-derived extracellular vesicles controls il-3 pro-angiogenic paracrine action



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ACTIVATED STAT5 TRAFFICKING VIA ENDOTHELIAL CELL-DERIVED EXTRACELLULAR VESICLES CONTROLS IL-3 PRO-ANGIOGENIC PARACRINE ACTION.

Giusy Lombardo, Patrizia Dentelli, Gabriele Togliatto, Arturo Rosso, Maddalena Gili, Sara Gallo, Maria Chiara Deregibus, Giovanni Camussi and Maria Felice Brizzi



Supplementary Information

Supplementary Figure S1. (a) Representative transmission electron microscopy (TEM) imaging of EVs negatively stained with NanoVan. EVs were viewed by JEOL Jem 1010 electron microscope (black line= 100 nm). (b)To evaluate EVs up-take by ECs, ECs were treated for 1h with PKH26-labeled IL-3-EVs and analyzed. Representative sections (first-middle-last) of images (Z-stack) obtained on a confocal microscope are reported. Four different experiments performed in triplicate (n=4). Scale bars indicate 10 μm. (c) Representative bioanalyzer profile of small RNAs performed on EVs or IL-3-EVs, showing an enrichment of small RNAs of the miRNA size.




Supplementary Figure S2. Representative FACS analysis of different EC-derived EVs reported in the histograms as the percentage of CD81, CD105, c-Kit/CD117, CD31, KDR and CD146 expression. Isotype controls were included.




Supplementary Figure S3. Representative FACS analysis of different EC-derived EVs reported as dot plot analysis for CD81, CD105, c-Kit/CD117, CD31, KDR and CD146. Isotype controls were included.



Supplementary Figure S4. Representative FACS analysis of double immunostaining for CD81 and CD146 markers on IL-3-EVs reported as dot plot analysis. Isotype controls were included. The results are representative of four different experiments (n=4) performed in triplicate (percentage of positivity: CD81=27±3; CD146=46±5; CD81/CD146=0.12±0.1)


Supplementary Figure S5. Absence of IL-3 in IL-3-EVs. (a) PCR was performed on reversed mRNA extracted from the indicated EC-derived EVs (right panel a) or from IL-3- or EV-treated ECs (left panel a) to evaluate IL-3 mRNA. Total RNA from the MLA cell line of a gibbon was used as positive control for IL-3 amplification primers. (b) Protein extracts from the indicated EVs were subjected to SDS-PAGE to evaluate IL-3 content. 2ng of the human recombinant IL-3 was used as positive control. (c) An Elisa assay was performed to evaluate membrane bound IL-3 on EVs. All the results are representative of four different experiments (n=4) performed in triplicate.



Supplementary Figure S6. miR-126-5p and pre-miR-126 content in EC-derived EVs. (a) miR‐126-3p and miR-126-5p expression was evaluated by quantitative real-time PCR (qRT-PCR) on ECs, untreated (c) or treated with IL-3 (IL-3) in the presence or in the absence of the anti-IL3Ralpha blocking antibody (anti-IL-3R). Data normalized to RNU6B are representative of five different experiments performed in triplicate (n=5) (b) miR‐126-3p and miR-126-5p expression was evaluated as above on EVs recovered from ECs, treated as above (n=5) (p=0.01 IL-3-EVs vs EVs; p=0.04 IL-3-EVs vs anti-IL-3R-EVs for miR-126-3p: p=0.001 IL-3-EVs vs EVs; p=0.01 IL-3-EVs vs anti-IL-3R-EVs for miR-126-5p). (c) pre-miR‐126 expression was evaluated by qRT-PCR on EVs recovered from ECs, untreated or treated as above (n=5) (p<0.05 IL-3-EVs vs EVs and anti-IL-3R-EVs). (d) ECs incubated in the presence of 50 g/ml of -amanitin to inhibit EC transcription were stimulated or not with EVs or IL-3-EVs. EV-pre-miR-126 transfer was evaluated by q-RT-PCR. The difference in Ct values (Ct) between -amanitin-treated ECs alone or with the indicated EVs is reported (p=0.002). The results are representative of four different experiments (n=4) performed in triplicate (mean±SD).




Supplementary Figure S7. Loss of function experiments for miR-126-3p expression. (a-b) miR‐126-3p expression was evaluated by qRT‐PCR on ECs that had either undergone transfection with anti-miR-126-3p oligonucleotides or not (a). EVs were isolated from these cells and analyzed by qRT-PCR for their miR-126-3p content (b). Data, normalized to RNU6B, are representative of four different experiments (n=4), performed in triplicate. (***p<0.001 anti-miR-126-3p transfected ECs, alone or plus IL-3, vs anti-miR neg in a; *p<0.05 IL-3-EVs anti-miR- neg vs EVs anti-miR- neg, **p<0.01 EVs and IL-3-EVs anti-miR-126-3p vs EVs and IL-3-EVs anti-miR neg in b)




Supplementary Figure S8. (a) Representative images of NanoSight analyses performed on the 100k fraction of EVs recovered from ECs untreated, IL-3-treated or transfected with NSTAT5 or 1*6STAT5 constructs. The JAK2 inhibitor was also used in ECs treated with IL-3. Curve 1, relationship between particle distribution (left y axis) and particle size (x axis); curve 2, correlation between cumulative percentage distribution of particles (percentile in right y axis) and particle size (x axis). (b) ECs treated with IL-3 alone or IL-3 plus the anti-JAK2 inhibitor were analyzed for pSTAT5 content and normalized to STAT5 (n=4); (**p<0.01 none and JAK2 inhibitor + IL-3 vs IL-3). (c) Number of EV particles (mean±SEM) calculated per cell at isolation. Data refer to EVs from ECs (EVs), IL-3-treated ECs (IL-3-EVs) or from IL-3-treated ECs plus JAK2 inhibitor (IL-3-iJAK2-EVs). The results are representative of four different experiments performed in triplicate (n=4) (*p<0.05, IL-3-iJAK2-EVs vs IL-3-EVs; **p<0.01 EVs vs IL-3-EVs).


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