GPCR activation in U2OS cells by selective agonists

(A) Dose-dependent cAMP accumulation after ADORA1 activation with agonist, CCPA, or inhibition with antagonist DPCPX (5μM). (B) Dose-dependent IP1 accumulation after PEA stimulation of HRH1 and inhibition by Cetirizine (1μM). (C) ALP activity after stimulation of FZD4 by Wnt3a. (D) The ratio of phosphorylated ERK1/2 (pERK1/2) by total ERK 1/2 (tERK1/2) after stimulation of ACKR3 by SDF-1α. For all graphs, data are shown as mean ± SD, with n=3 independent repeats, each having duplicate determinants, ns=not significant, *p < 0.05, **p < 0.01, ***p < 0.001 vs vehicle control (VC). Statistical significances were determined by one way or two-way ANOVA of receptors by agonists or antagonists and post-hoc Tukey or Sidak testing for multiple comparisons.

Characterization of EVs isolated from U2OS cell culture media

(A) Representative immunoblots of EVs isolated by SEC from U2OS cell culture media (U2) but not from media control (MC) show detection of EV markers including CD9, CD63, CD81, flotillin, and syntenin, in EV fractions 7-9. Endoplasmic reticulum marker, calnexin, was not detected in the EV fractions. (B) Representative images from transmission electron microscopy of isolated EVs (red arrows) from pooled EV-enriched fractions 7-9. (C) Size distribution of pooled EV fractions 7-9 isolated from MC and U2. (D) Quantification of pooled EV fractions 7-9 of MC and U2 measured by f-NTA. Data are shown as mean ± SEM, n=7-8. A Student’s t-test determined significant differences in the EV concentration between the MC and U2, *p < 0.05.

Differentially expressed EV miRNAs in response to GPCR activation

(A) The heatmap showed unsupervised hierarchical clustering of miRNA expression (row) following GPCR stimulation (column). The relative abundance of miRNAs is represented in Z-score value (z-transformed fold changes), blue, below-mean expression; red, above-mean expression. (B-E) Volcano plots displayed the analysis of EV miRNAs after ADORA1 (B), HRH1 (C), FZD4 (D), and ACKR3 (E) activation. On the x-axis, the dotted line indicates miRNAs that satisfied the |log2 fold change|≥ 1.5 cut-off, while the dotted line on the y-axis indicates miRNA that met Skillings-Mack p-value < 0.2. (F) Venn diagram showed the miRNAs of interest with at least 1.5-fold change across four GPCR groups following treatment. n=5-6 replicates per GPCR group.

EV miRNAs differentially expressed in 4 GPCR groups.

Differentially expressed miRNAs (p < 0.05) were identified in each treatment group compared to its vehicle control, n=5-6 samples of each group.

Pathway analysis of the differentially expressed EV miRNA after GPCR activation

The bubble plots showed the top 25 significantly enriched KEGG pathways for the miRNAs (≥ 1.5-fold change) of individual GPCR. (A) ADORA1, (B) HRH1, (C) FZD4, (D) ACKR3. The dot size represents the number of enriched gene targets, and the color shows the p-value of the enrichment. For the enrichment analysis, cut-off criteria were p-value (FDR) < 0.05 and gene count > 2. KEGG terms: * Endocrine and other factor-regulated calcium reabsorption, ^ Progesterone-mediated oocyte maturation.