HDAC inhibitors tune miRNAs in Extracellular Vesicles of Dystrophic Muscle-Resident Mesenchymal Cells (2020)
Figure 5

Fig. 5: EVs from FAPs in vivo exposed to TSA promote MuSCs differentiation, expansion and asymmetric division.

A) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). Nuclei were counterstained with DAPI (blue). MuSCs were cultured alone (-) or with EVs (EVs FAPs) isolated from FAPs in vivo exposed or not to TSA (CTR and TSA) and transfected or not with Antagomir-206 (- and A-206). (n=7 for all samples, but antagomir's samples n=3, biological replicates). Scale bar =50 μm.

(B) Graph showing the fusion index of MuSCs in the condition described in (A). Star (*) indicates statistical analysis by Tukey test relative to MuSCs cultured alone (-), *p < 0.05, ****p>0,0001; hash (#) means significance compared to EVs-FAPs CTR -, ###p<0,001; and (†) means significance to EV-FAPs TSA -, †††† p<0,0001. § indicates significance by Anova test; §§§§ p>0,0001.

(C) Graph relative to Notch3 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) (n=3). Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR, ##p<0,01. § indicates significance by Anova test; §§ p>0,01.

(D) Graph relative to Notch1 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) n=3. Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR; *p < 0.05. § indicates significance by Anova test; §§ p>0,01.

(E) Representative images showing myofibers cultured alone (Ctr) or with extracellular vesicles isolated from FAPs (EVs-FAPs) and stained with PKH-67 (green) and MyoD (red). Nuclei were counterstained with DAPI (blue). Arrows represent nuclei positive for both PKH-67 and MyoD. Scale bar = 25 μm.

(F) From left to right: representative images of myofibers cultured for 24 hours alone (-) or with conditioned media of FAPs that were previously exposed or not to TSA and GW4869 (MEDIA-FAPs) or extracellular vesicles (EVs-FAPs) collected from FAPs exposed or not to TSA in vivo (CTR and TSA) and transfected with antagomiR-206 (TSA A-206). Myofibers have been stained for EdU(Cyan), immunofluorescence with Pax7 (green), MyoD (red). Nuclei were counterstained with DAPI (blue). (n=3). Scale bar = 25 μm.

(G) Graph showing the average of nuclei positive for Pax7 (green), MyoD (red) in the conditions indicated in (F). Star (*) indicates statistical analysis by Tukey test relative to myofibers cultured alone (-), (n=3, biological replicates). *p<0,05, **p< 0.01; ***p<0,001; ***p<0,0001; hash (#) means significance compared to MEDIA FAPs CTR, #p<0,05; (†) means significance to EV-FAPs CTR, † p<0,05; while ¶ means significance compare to EVs-FAPs TSA, ¶¶¶¶ p<0,0001. § means significance by Anova test; §§ p < 0.01.

Data information: All data correspond to the average ± SEM.

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