Fig. 7.

CircFndc3b-FUS-VEGF-A signaling network regulates cardiac endothelial cell function: a RNA binding protein immunoprecipitation analysis was carried out using anti-FUS or AGO2 or IgG control antibodies. Total RNA was isolated and digested with or without RNase R, and b circFndc3b levels in the samples was quantified using RT-qPCR, normalized to 18S rRNA. n = 5 independent experiments per group. Data were Mean ± SEM.*p < 0.05, **p < 0.01 vs IgG (one-way ANOVA); c Representative western blot images of FUS and VEGF-A protein levels in MCECs treated with control or circFndc3b overexpression plasmids; d, e Quantification of FUS and VEGF-A protein levels normalized to β-actin. Data were Mean ± SEM of three independent experiments. *p < 0.05 **p < 0.01 vs control plasmid (one-way ANOVA); f MCECs were transfected alone or in combination with FUS siRNA, FUS overexpression plasmid, or circFndc3b overexpression plasmid or their respective controls for 48 h. f Representative western blot images of VEGF-A; g Quantification of VEGF-A levels normalized to β-actin. Data shown are Mean ± SEM of 3 independent experiments **p < 0.05 vs control plasmid, ^##p < 0.01 vs circFndc3b overexpressing cells. ^p < 0.05 vs FUS OE + circFndc3b OE treated cells (one-way ANOVA). h Quantification of TUNEL + MCECs presented as the % TUNEL + positive cells and DAPI-stained nuclei MCECs subjected to H₂O₂ (100 μM) stress. Data shown are Mean ± SEM of n = 3–7/group. **p < 0.01 ***p < 0.001 vs control plasmid, ^###p < 0.001 vs circFndc3b treated cells, ^$$$p < 0.001 vs FUS OE treated cells, ^p < 0.05 vs FUS OE + circFndc3b treated cells (one-way ANOVA). i MCECs were treated as above and trans-well migration assay was performed. Quantification of migrated cells presented as migrated cells counted by DAPI-stained nuclei. Data shown are Mean ± SEM of n = 3–7 independent experiments. *p < 0.05 vs control plasmid, ^##p < 0.01 vs circFndc3b treated cells, ^$p < 0.05 vs FUS OE treated cells, ^p < 0.05 ^^^p < 0.001 vs FUS OE + circFndc3b treated cells (one-way ANOVA). j RT-qPCR of FUS expression in LV tissue of AAV9 control (n = 4 mice) or AAV9 circFndc3b (n = 5 mice) treated mouse hearts at 8 weeks post-MI. Data were normalized using 18S rRNA. Data shown are Mean ± SEM. *p < 0.05 vs AAV9 control or saline treated hearts (two-sided unpaired students t-test); k, l Representative western blot images of VEGF-A and its quantification in LV tissue of AAV9 control or AAV9 circFndc3b treated hearts at 8 weeks post-MI. Data was normalized using β-actin. n = 4 mice/group. *p < 0.05 vs AAV9 control or saline treated hearts (two-sided unpaired students t-test)