Fig. 3: CircFNDC3B interacts with TFIII in NSCLC cells.

A RIP assay with anti-AGO2 or IgG antibodies. U6 and CDR1as were used as negative and positive controls, respectively. Data were displayed with mean ± SD. (Student’s t-test, n = 3, ns, not significant, ***P < 0.001). B Biotin-labeled antisense or sense circFNDC3B probes were applied for RNA-protein pull-down assay using A549 cell lysates. The silver staining was used to distinguish the proteins interacting with circFNDC3B. The primary differential band precipitated in A549 lysates was indicated by red arrow. C The TFII-I peptides pulled down by sense circFNDC3B probes were demonstrated by MS. D The interactions between circFNDC3B and TFII-I or AGO2 were detected by RNA pull-down assay and western blot assay in A549 and H226 cells. The loading control was represented with GAPDH. E Purified TFII-I protein was preincubated with lysis of A549 and H226 cells. RIP experiments were performed with IgG and TFII-I antibodies. Utilized the antibodies against TFII-I, the precipitates were subjected to western blot assay. The relative levels of TFII-I-enriched or Input-enriched and IgG-enriched circFNDC3B were determined by RT-qPCR. Data were displayed as mean ± SD. (Student’s t-test, n = 3, **P < 0.01, ***P < 0.001). F Colocalization of circFNDC3B (red) and TFII-I (green) in A549 and H226 cells was detected by confocal microscopy. Nuclei were stained with DAPI. G Schematic diagram displayed full-length and truncated TFII-I protein. H, I Flag-tagged domain truncated or full-length TFII-I RIP assays in A549 cells. Using anti-Flag antibody, the precipitate was subjected to western blot assay (H). Relative enrichment of endogenous circFNDC3B was analyzed by RT-qPCR (I). Data were displayed as mean ± SD. (Student’s t-test, n = 3, ns, not significant, **P < 0.01).