Fig 1: 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.
Fig 2: USP18 is up-regulated in bladder cancer and potentially associated with FTO protein level. (A) Schematic of the putative ubiquitination sites of K194, K211 and K216 (rhombus in green) in the N-terminal domain (NTD) of FTO as predicted by PTMcode tool. (B) Venn diagram showing FTO-targeting ubiquitin ligases and deubiquitinases that are significantly upregulated in BLCA. (C) Diagram showing the physical interaction between FTO and USP18 as predicated by HDOCK server. (D) USP18 transcript in patient-derived bladder tumor (T) versus normal tissues (N). *** p < 0.001, student’s t-test. (E) USP18 protein in BLCA tumor (T) versus normal tissues (N) as determined by western blot assay. ** p < 0.01, student’s t-test. (F) Pearson correlation analysis of FTO with USP18 expression. (G) USP18 protein expression profiling in the representative BLCA cell lines.
Fig 3: USP18 is upregulated in human cervical cancer tissues. a USP18 is upregulated in primary cervical cancer samples. Data collected from the TCGA database CESC dataset. *** p < 0.001 vs Normal. b The relative mRNA levels of USP18 were much higher in human cervical cancer tissues than that in para-cancer tissues, n = 30 for each group. *** p < 0.001 vs Normal. c USP18 was enriched in the PI3/AKT pathway. d The relative protein levels of USP18 and AKT phosphorylation were upregulated in human cervical cancer tissues compared with that in para-cancer tissues. *** p < 0.001 vs para-carcinoma tissues. n = 4 for each group. The full-length gels are presented in Supplementary Figure 1D. e IHC staining assay indicated the positive correlation between USP18 and AKT phosphorylation in human cervical cancer tissues, n = 30 for each group
Fig 4: USP18 imposes post-translational deubiquitination on FTO. (A, B) Determining USP18, FTO mRNA (A) and protein (B) expression upon USP18 depletion. USP18 was knocked down by transfecting USP18 siRNA-248 (si-USP-1) and USP18 siRNA-581 (si-USP-2). NC, negative control transfected with scramble shRNA. *** p < 0.001, student’s t-test. n.s., non-significant. (C) Co-immunoprecipitation determining the direct interaction between FTO and USP18. NC, negative control; OE, overexpression. (D) Co-immunoprecipitation of FTO protein and ubiquitin in the presence or absence of USP18. USP18 was knocked down by transfecting USP18 siRNA-248. (E) Western blot determining FTO protein stability in the presence or absence of USP18. USP18 was knocked down by transfecting USP18 siRNA-248. (F) Western blot showing that blockage of proteasomal degradation with MG132 stabilized FTO protein upon depletion of USP18. USP18 was knocked down by transfecting USP18 siRNA-248.
Fig 5: USP18 silencing suppressed the proliferation and induced apoptosis in human cervical cancer cells. a & b siUSP18–1 or siUSP18–2 significantly suppressed the proliferation of Caski and SiHa cells, n = 3 for each group. * p < 0.05 vs siNC; *** p < 0.001 vs siNC. c The apoptosis of Caski and SiHa cells were upregulated after transfecting with siUSP18–1 or siUSP18–2, n = 3 for each group. *** p < 0.001 vs siNC. d & e Western blot was used to examine the protein contents of Ki-67, Cyclin D1, cleaved PARP, BAX and ß-catenin in Caski and SiHa cells that transfected with siUSP18–1 or siUSP18–2 respectively, n = 3 for each group. *** p < 0.001 vs siNC. The full-length gels are presented in Supplementary Figure 3D-E. f & g Western blot was used to examine the protein contents of USP18, Cleaved caspase-3, AKT and p-AKT in Caski and SiHa cells that transfected with siUSP18–1 or siUSP18–2 respectively, n = 3 for each group. *** p < 0.001 vs siNC. The full-length gels are presented in Supplementary Figure 3F-G
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