Figure 4
Figure 4. Gene expression analysis of exosomal miR-214 effects on recipient endothelial cells. (A) Gene expression analysis was performed on endothelial cells exposed to control miR-214 or anti–miR-214 exosomes relative to endothelial cells that were incubated in the absence of purified exosomes and are presented in a heat map displaying genes that were upregulated (blue) or downregulated (yellow). (B) The list identifies the top 10 downregulated and upregulated probes in recipient cells treated with anti–miR-214 exosomes compared with cells subjected to control miR-214 exosomes. (C) The list identifies the top 5 biological processes (with accompanying GO terms) in recipient cells regulated by exosomal miR-214. (D) The predicted miR-214 seed sequence in the ATM 3′UTR region is shown. (E) The graph shows the results of miR-214 ATM luciferase reporter assay (n = 3, Student t test). (F) ATM protein levels in lysates from cells incubated either without (basal) or with control miR-214 (ctrl) or anti–miR-214 exosomes (anti) were determined by immunoblotting (upper panel; T, top of gel; 170 indicates MW of the highest MW marker band), and quantitation using β-actin was used as a loading control (n = 3, ± SD, ANOVA). (G) Quantitation of a migration assay with cells transfected with control or ATM-siRNA is shown (n = 3, ± SD, Student t test). (H) β-galactosidase staining (upper panel) was used to quantify the percentage of senescent cells (lower panel; n = 4, ± SD, ANOVA).

Gene expression analysis of exosomal miR-214 effects on recipient endothelial cells. (A) Gene expression analysis was performed on endothelial cells exposed to control miR-214 or anti–miR-214 exosomes relative to endothelial cells that were incubated in the absence of purified exosomes and are presented in a heat map displaying genes that were upregulated (blue) or downregulated (yellow). (B) The list identifies the top 10 downregulated and upregulated probes in recipient cells treated with anti–miR-214 exosomes compared with cells subjected to control miR-214 exosomes. (C) The list identifies the top 5 biological processes (with accompanying GO terms) in recipient cells regulated by exosomal miR-214. (D) The predicted miR-214 seed sequence in the ATM 3′UTR region is shown. (E) The graph shows the results of miR-214 ATM luciferase reporter assay (n = 3, Student t test). (F) ATM protein levels in lysates from cells incubated either without (basal) or with control miR-214 (ctrl) or anti–miR-214 exosomes (anti) were determined by immunoblotting (upper panel; T, top of gel; 170 indicates MW of the highest MW marker band), and quantitation using β-actin was used as a loading control (n = 3, ± SD, ANOVA). (G) Quantitation of a migration assay with cells transfected with control or ATM-siRNA is shown (n = 3, ± SD, Student t test). (H) β-galactosidase staining (upper panel) was used to quantify the percentage of senescent cells (lower panel; n = 4, ± SD, ANOVA).

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