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. Author manuscript; available in PMC: 2022 Jun 4.
Published in final edited form as: Nat Aging. 2021 Dec 6;1(12):1148–1161. doi: 10.1038/s43587-021-00143-2

Fig. 5. Klotho transcripts in EVs decline over time and are preferentially contained within EVs with high expression of the CD81 surface marker.

Fig. 5.

a, Imaging and b, quantification of Klotho (green) in MPCs cultured in the presence or absence of young EVs in culturing media. Scale bar: 50 μm. (*p<0.05, **p<0.01, one-way ANOVA with Tukey’s multiple comparisons, n= 6 (Young MPCs), 7 (No EVs), 7 (Young serum EVs) wells). c, Quantification of secreted Klotho in conditioned media of aged MPCs treated with or without young EVs (****p < 0.01, two-tailed Mann Whitney test, n=11 (No EVs), 20 (Young serum EVs)). d, Klotho quantification in Klotho−/− MPCs in the presence of no EVs or young EVs (****p<0.0001, two-tailed Welch’s t-test, n=6 wells/group). e, Quantification of secreted Klotho in culture media of Klotho−/− MPCs in the presence or absence of young EVs (***p<0.001, two-tailed Welch’s test, n=14 (No EVs), 17 (Young serum EVs)). f, Surface Plasmon Resonance imaging (SPRi) and analysis of Klotho protein in young and aged serum EVs. (p>0.05, two-tailed Student’s t test, n=4 (Young), 7(Aged)). g, Rug plot with beeswarm plot of Klotho mRNAs in young and aged serum EVs. h, 3D-plots of Klotho mRNA distribution in CD63 and CD81 positive circulating EVs. i, Klotho mRNAs quantification in young and aged human serum EVs. (p>0.05, two-tailed Welch’s t-test, n=5/group) j, Human EV-Klotho mRNA quantification in publicly archived dataset (n=32). k, Klotho quantification in aged MPCs receiving young EVs treated with either non-targeting siRNA (scramble) or siRNA to Klotho (***p<0.001, two-tailed Welch’s t-test, n=6 wells/group, repeated in two independent experiments). l, Klotho quantification in Kl−/− MPCs receiving young EVs treated either with a non-targeting siRNA (scramble) or siRNA to Klotho (***p<0.001, two-tailed Welch’s t-test, n=6 wells/group, repeated in two independent experiments). m, Imaging and quantification of Klotho protein (green) in aged MPCs receiving aged EVs engineered with synthetic Klotho mRNA (pink). Scale: 50 μm. (*p<0.05, two-tailed Welch’s t-test, n=12 (aged EVs), 14 (aged EVs+synKL) wells, presented as fold change over aged serum EVs per experiment). n, Klotho quantification in conditioned media of aged MPCs administered with aged EVs or aged EVs engineered with synthetic Klotho mRNA (**p<0.01, two-tailed Student’ t test, n=5 (aged EVs), 6 (aged EVs+synKL)). o, Cardiolipin (NAO) quantification in aged MPCs treated with aged EVs or aged EVs engineered with synthetic mRNA (**p<0.01, two-tailed Mann Whitney test, n=3 wells/group, data representative of one of the two independent experiments). Data presented as mean ± SEM and different cohorts performed on different days presented within the same graph as black, red, or blue circles.