Fig 1: EDD siRNA abrogates nocodazole-mediated G2/M accumulation and increases expression of CDC20, phospho-APC3, and BUBR1. HeLa cells were treated with the indicated siRNAs, treated with nocodazole (A–C, E, and F) or Taxol (D) for 18 h, released, and imaged by brightfield microscopy (A) and analyzed by propidium iodide staining and FACS analysis (B and D, respectively). siRNA efficiency (C) and siRNA-treated cells released from Noc for the indicated times post-release (E) were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. F, density quantification of bands in E, with the values expressed relative to scrambled siRNA t0 values. The data in B and D were analyzed using one-way analysis of variance and Tukey's multiple comparison test, which revealed all G1 or G2/M comparisons to be significantly different.
Fig 2: Generation of Fzr19A/9A knockin mice. (A)Schematic representation of the WT Fzr1+ allele, the Gene trapped (GT) allele Fzr1GT, the knock-in (KI) alleles of Fzr1GT wt and Fzr19A and the replacement vectors possessing Fzr1GT wt or Fzr19A cDNA. Fzr1 GT allele was generated by integration of the exchangeable Gene Trap (GT) vector in the 1st intron of the endogenous Fzr1 locus30. Cre-mediated recombination between Fzr1GT allele and the replacement vector generated Fzr19A KI allele. The control Fzr1wt allele was generated between Fzr1GT allele and the replacement vector possessing Fzr1 wt cDNA in the same manner. SA: splicing acceptor, Frt: Flippase recombination site, polyA: poly adenylation signal, lox P and its variant recombination sites are indicated by triangles. Light blue rectangles indicate the exons of Fzr1 locus. Arrows indicate the PCR primers for genotyping. (B) Nine putative CDK-phosphorylated sites of Ser and Thr residues in FZR1, where Ala substitutions were introduced. (C) PCR genotyping of genomic DNA from Fzr1+/+, Fzr1+/9A, Fzr19A/9A and Fzr1Gt wt/Gt wt KI mice. (D) Western blot of testis extracts from Fzr1+/+, Fzr1Gt wt/Gt wt and Fzr19A/9A testes (P18), probed by antibodies as indicated. (E) Western blot of immunoprecipitates from testis extracts of Fzr1+/+, Fzr1Gt wt/Gt wt and Fzr19A/9A mice using anti-CDC27 antibody. *Indicates nonspecific bands cross-reacted with IgG. See also Supplementary Fig. S2 for the uncropped images.
Fig 3: Representative images and analysis of CDC27 immunohistochemistry staining from the tissue microarray of specimens from patients with READ. (A) Expression of CDC27 in the nucleus and/or cytoplasm of tumor cells. Scale bars, 50 µm (magnification, ×40) and 20 µm (magnification, ×10). (B) Distribution of CDC27 expression in the cytoplasm and nucleus by the H-index. Data are expressed as the mean ± SD. (C) The correlation of CDC27 expression between the cytoplasm and nucleus as determined using Spearman's correlation analysis. CDC27, cell division cycle 27.
Fig 4: Cell cycle degradation of Mcm10 is independent of APC. (A) HeLa cells were transfected on three consecutive days with APC3 or control GL2 siRNA. After the third transfection, the cells were arrested with nocodazole for 15 h and were harvested after release from nocodazole (Noc) for analysis of Mcm10 protein, along with asynchronous cells (ASN). Samples were also taken 2 h after release from nocodazole (2 h) (B) The decrease of APC3 protein after RNAi was confirmed by immunoblotting with anti-APC3 antibody. NS points to a non-specific band that displays equal protein loading in different lanes. (C) The decrease of APC3 mRNA was confirmed by RT-PCR and the numbers indicate the relative intensity of the mRNA. (D) Flow cytometry of propidium iodide-stained DNA of HeLa cells, as described in (A) confirms a M-phase block and release after siRNA depletion. The observed mobility of endogenous APC3 was 73 kDa.
Fig 5: AGPG affects PFKFB3 stability by preventing its ubiquitination.a In vitro-synthesized AGPG was incubated with protein lysates from KYSE30 cells transfected with vectors expressing FLAG-tagged FL or truncation mutants of PFKFB3. RNA pull-down and western blotting assays were then performed. Truncation mutants included FL, N-terminal (N) and C-terminal (C) constructs. b RIP assays were performed using anti-FLAG antibodies in cells transfected with vectors expressing FLAG-tagged FL or truncation mutants of PFKFB3. c AGPG knockdown reduced PFKFB3 expression in ESCC cells. d PFKFB3 downregulation by AGPG CRISPR KO was rescued by AGPG FL but not by AGPG ?T5. e PFKFB3 downregulation by AGPG knockdown was abolished by MG-132 (10 µm, 12 h). f Western blotting detection of PFKFB3 levels in KYSE150 cells transfected with shCtrl or shAGPG followed by treatment with CHX (100 µg per ml) for the indicated times. g IP assays showed that AGPG knockdown increased PFKFB3 ubiquitination levels. FLAG-tagged PFKFB3 was expressed in cells, which were then subjected to IP assays. h Active APC/C could be immunoprecipitated from cells using monoclonal Cdc27 antibody. CoIP assays showed that AGPG CRISPR KO significantly increased the interaction between PFKFB3 and Cdc27. i IP assays showed that AGPG knockdown did not increase ubiquitination of the PFKFB3 K302A mutant. j Cells were infected with FLAG-tagged PFKFB3 WT or K302A and treated with CHX (100 µg per ml) for the indicated time. FLAG levels were detected by western blotting. k PFKFB3 K302A overexpression significantly reversed the decreased ECAR and cell proliferation caused by AGPG CRISPR KO, whereas PFKFB3 WT could only partially rescue these effects in KYSE150 cells. l PFKFB3 K302A overexpression significantly reversed the decreased glycolysis caused by AGPG CRISPR KO, whereas PFKFB3 WT could only partially rescue this effect. m PFKFB3 K302A overexpression abolished the G1/S arrest caused by AGPG CRISPR KO, whereas PFKFB3 WT could only partially rescue this effect. Data in b, k–m are representative of three independent experiments and presented as mean±S.D., n = 3 biologically independent samples, the P value was determined by one-way ANOVA with Tukey’s multiple comparisons test. No adjustments were made for multiple comparisons.
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