Fig. 7

Exosomes could deliver circNEIL3 to TAMs, thereby enabling them to acquire angiogenic and immunosuppressive properties. A RIP and qRT-PCR assays showing the interaction brtween circNEIL3 and hnRNPA2B1, using Igg and hnRNPA2B1 antibodies, n = 3. B RNA pulldown and western blot assays showing the interaction brtween circNEIL3 and hnRNPA2B1. qRT-PCR assay showing the relative expression in (left) GBM cells transfected with si-NC or si-hnRNPA2B1, and (right) exosomes collected from GBM cells transfected with si-NC or si-hnRNPA2B1, n = 3. D Representative flow cytometry assay showing the proportion of CD11b + CD163+ in THP1 differentiated macrophages transfected with ov-NC or ov-circNEIL3. Quantification histogram represented the proportion of CD11b + CD163+ in THP1 differentiated macrophages, n = 3. E Western blot assays showing the expression of SPP1 in THP1 differentiated macrophages (Left) transfected with ov-NC or ov-circNEIL3, and (Right) treated with exosomes collected from GBM cells transfected with si-circNEIL3, blank or ov-circNEIL3. F Representative flow cytometry assay showing the proportion of CD11b + CD163+ in THP1 differentiated macrophages treated with exosomes collected from GBM cells transfected with si-circNEIL3, blank or ov-circNEIL3. Quantification histogram represented the proportion of CD11b + CD163+ in THP1 differentiated macrophages, n = 3. G RNA pulldown and western blot assays showing the interaction between circNEIL3 and IGF2BP3 in THP1 differentiated macrophages in the indicated group. H RIP and qRT-PCR assays showing the interaction between circNEIL3 and hnRNPA2B1, using Igg and IGF2BP3 antibodies, n = 3. I Western blot assays showing the protein levels of IGF2BP3 in THP1 differentiated macrophages transfected with ov-NC or ov-circNEIL3, treated with 20 μg/ml CHX for the indicated periods of time. J Co-IP assay showing the ubiquitination modification level of IGF2BP3 in THP1 differentiated macrophages transfected with ov-NC or ov-circNEIL3. Western blot assays showing the protein levels of YAP1 in THP1 differentiated macrophages transfected with (K) left, ov-NC or ov-circNEIL3, and right, sh-NC or sh-circNEIL3 as indicated, and (L) left, ov-NC or ov-IGF2BP3, and right, sh-NC or sh-IGF2BP3 as indicated. M Western blot assays showing the YAP1 protein expression in THP1-differentiated macrophages co-transfected with ov-NC or ov-circNEIL3 and si-IGF2BP3 as indicated. N Left, representative bioluminescent image showing the tumor size for animals in different groups in the indicated time. n = 5 for each group. Right, statistical analysis of bioluminescent tracking plots. O Kaplan-Meier survival curves for animals in different groups, n = 5 for each group. P Represented H&E staining images for a subgroup of animal sacrificed simultaneously in each group, n = 5 for each group, scale bar, 10 μm. Q Proposed working model of circNEIL3 functions in tumorgenesis and malignant progression of glioma. All data are presented as the means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001