Fig 2: HECTD1 is crucial for the functional role of LMX1B in PCa. (a) HECTD1 expression in vector and circGFRA1 transfected cells measured by qRT-PCR and western blotting using the vector group as control. (b) HECTD1 expression in circGFRA1-si-NC, circGFRA1-si-HECTD1#1, and circGFRA1-si-HECTD1#2 transfected cells measured by qRT-PCR and western blotting. (c) Cell proliferation was detected by CCK-8. (d and e) EdU assay was performed to evaluate cell proliferation. (f and g) Cell migration assessed by the transwell assay. (h) VEGF content of PCa cell supernatants. (i) IL-10 content of PCa cell supernatants. (j) TGF-ß1 content of PCa cell supernatants. (k and l) The cytotoxicity of CIK cells to PC-3 and LNCap cells was determined via CCK-8 assay using the circGFRA1-si-NC group as control. Data are presented as mean ± standard error. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: LMX1B binds to circGFRA1 and upregulates circGFRA1 expression in PCa cells. (a) Relative circGFRA1 expression in PC-3 and LNCap cells upon 0/20/50/100 ng LMX1B transfection and circGFRA1 expression upon 0 ng LMX1B transfection were used as control. (b) Relative circGFRA1 expression in PCa cells upon si-NC, si-LMX1B#1, and si-LMX1B#2 transfection results from si-NC group was used as control. (c) The predicted binding motif of LMX1B obtained from JASPAR. (d) The predicted binding motif of GFRA1 promoter regions obtained from JASPAR. (e and f) ChIP analysis of enrichment of LMX1B on the GFRA1 promoter in PC-3 and LNCap cells. IgG was used as a negative control. (g and h) Quantification of the luciferase activity of the wild-type or mutant GFRA1 promoter reporter in PC-3 and LNCap cells. Data are presented as mean ± standard error. *P < 0.05, ***P < 0.001.