Fig. 4

CircNEIL3 physically interacts with IGF2BP3 and inhibits its ubiquitination. A Different protein bands detected by silver stain for mass spectrometry of the circNEIL3-protein complex pulled down by circNEIL3 junction sense or anti-sense in U251 cells, the arrow points to IGF2BP3 band. B RNA pull down and western blot assays showing the interaction between IGF2BP3 with circNEIL3 in U251 and A172 cells. GAPDH was used as a negative control. C RIP and qRT-PCR assays showing the interaction between IGF2BP3 with circNEIL3 in GBM cells, using Igg and IGF2BP3 antibodies, n = 3. D IF-Fish assay showing co-localization of circNEIL3 (red) with IGFB2P3 proteins (green) in U251 and A172 cells. Scale bar, 25 μm. E Schematic structures of IGF2BP3 proteins and five mutants (Delet KH1–2, Delet KH3–4, RRM 1–2, KH1–2, KH3–4). F Western blot assay showing the full length of IGF2BP3 and its mutants. G RIP and qRT-PCR assays showed that the KH3–4 fragment of IGF2BP3 was the region responsible for circNEIL3 binding. H RNA pull-down and western blot assays showed that the interaction site on circNEIL3 with IGF2BP3. I The mRNA expression of IGF2BP3 was detected by qRT-PCR in GBM cells transfected with ov-NC or ov-circNEIL3, n = 3. J Western blot assays showing the protein levels of IGF2BP3 and downstream genes, including CD44, CDK4, CDK6 and c-MYC in GBM cells transfected with si-NC or si-circNEIL3, and ov-NC or ov-circNEIL3. K Western blot assays showing the protein levels of IGF2BP3 in GBM cells transfected with ov-NC or ov-circNEIL3, treated with 20 μg/ml CHX for the indicated periods of time. L Co-IP assay showing the ubiquitination modification level of IGF2BP3 in GBM cells transfected with ov-NC or ov-circNEIL3. Ub ubiquitin. M Co-IP assay showing the ubiquitination modification level of IGF2BP3 in GBM cells co-transfected with ov-NC or ov-circNEIL3 and vetors expressing FLAG-tagged WT or IGF2BP3 mutants (K450) as indicated. N Conservation ability of the K405 ub site on IGF2BP3 protein. O Crystal structure of IGF2BP3 proteins with K405. All data are presented as the means ± SD, ns, P > 0.05, ***p < 0.001