Research Paper Volume 13, Issue 3 pp 3909—3925

Dysregulation of USP18/FTO/PYCR1 signaling network promotes bladder cancer development and progression

FTO reduces m6A methylation on PYCR1 and enhances its RNA stability. (A, B) Determining PYCR1 mRNA (A) and protein (B) expression upon gain (FTOOE) or loss (siFTO) of FTO. * p t-test. (C) Co-immunoprecipitation of PYCR1 protein and ubiquitin in the presence or absence of USP18. (D) M6A dot blot (upper) and methylene blue staining (lower) assays determining effects of FTO in the total RNA m6A contents in BLCA cells. DOE, FTO and METLL3 double overexpression. MB, methylene blue. (E) M6A-IP-qPCR assay determining the effects of FTO in PYCR1 m6A methylation. DOE, FTO and METLL3 double overexpression. (F) qPCR determining PYCR1 RNA stability upon gain or loss of FTO in BLCA cells. 18S RNA was used as a normalization control.

Figure 6. FTO reduces m6A methylation on PYCR1 and enhances its RNA stability. (A, B) Determining PYCR1 mRNA (A) and protein (B) expression upon gain (FTOOE) or loss (siFTO) of FTO. * p < 0.05, ** p < 0.01, *** p < 0.001 versus NC group, student’s t-test. (C) Co-immunoprecipitation of PYCR1 protein and ubiquitin in the presence or absence of USP18. (D) M6A dot blot (upper) and methylene blue staining (lower) assays determining effects of FTO in the total RNA m6A contents in BLCA cells. DOE, FTO and METLL3 double overexpression. MB, methylene blue. (E) M6A-IP-qPCR assay determining the effects of FTO in PYCR1 m6A methylation. DOE, FTO and METLL3 double overexpression. (F) qPCR determining PYCR1 RNA stability upon gain or loss of FTO in BLCA cells. 18S RNA was used as a normalization control.