Fig 2: USP22 directly deubiquitylates CCND1 protein independent of phosphorylation at T286. (A) H1299 cell lysates subjected to an endogenous CCND1 IP under nondenaturing conditions using A/G beads. Precipitates probed for CCND1 to detect high molecular-weight species, presumably ubiquitylated CCND1. (B) Sf9 cells were infected with baculovirus-expressing Flag-Fbxo4, Flag-cyclin D1, CDK4, HA-a-B-crystallin, HA-Cul1, HA-Skp1, and HA-Rbx1. SCFFbxo4 complex and CCND1/CDK4 complex then purified using anti-FLAG M2 and combined with E1/E2 ligases, ATP, and ubiquitin for 30 min at 37 °C to ubiquitylate CCND1 protein. Postubiquitylation assay, the CCND1/CDK4 complex was isolated and incubated with or without USP22 at 37 °C for indicated times in deubiquitylation buffer. Protein levels detected by IB; high molecular-weight CCND1 species indicative of polyubiquitylated CCND1. (C) HCT116 cell lysates subjected to in vitro analysis of CCND1 ubiquitylation status following USP22 depletion using UbiTest, as described in the Materials and Methods. Cells treated with MG132 before harvest and lysates generated using buffer containing protease mixture inhibitor, pan DUB inhibitor PR619, and the conventional chelator o-phenanthroline. Eluates then either left undigested (lanes 1 and 2) or subsequently digested with a global-DUB to strip polyubiquitin (lanes 3 and 4). Protein levels were detected by IB. The black arrowheads indicate unit length CCND1 (nonubiquitylated) and the green arrowheads indicate high molecular-weight species presumably representing ubiquitylated CCND1. (D) Schematic representing UbiTest experimental design from C. (E) H1299 cells made to express HA-tagged CCND1 WT or mutant with lysines from K33-K123. USP22 depleted in cells with shRNA-mediated lentivirus and CCND1 protein levels were assessed via anti-HA by IB. (F) HA-tagged CCND1 was expressed in H1299 cells as either WT or with phosphorylation site mutation T286A. Following USP22 depletion with two distinct shRNA constructs, cells were lysed and CCND1 protein levels were assessed by IB. (G) H1299 cells expressing the WT or T286A isoforms of HA-tagged CCND1 were subjected to USP22 depletion and subsequent analysis by IB in the presence or absence of proteasome inhibition (EPX). (H) Phosphorylation-dependence of USP22-depletion induced CCND1 degradation was assessed by inhibition of the T286 kinase GSK3ß via treatment with TSW-119. Following depletion of USP22, CCND1 protein levels were assessed by IB. The established GSK3ß pathway substrate ß-catenin was included as control for TSW119 efficacy.

Fig 3: A putative NLS does not contribute to Ank13 nucleotropism. (A) Schematics of wild-type and Ank13 truncated mutant proteins. A red hatched line box indicates the putative NLS that consists of residues 10 to 42 and occurs within ankyrin repeat 1. (B) Confirmation that Ank13ΔF-box fails to interact with SCF ubiquitin ligase components. HeLa cells were transfected to express Flag-tagged Ank13, Ank1349-490, or Ank13ΔF-box. Input lysates were subjected to Western blotting with Flag antibody to verify ectopic expression of the proteins of interest; antibodies against Skp1, Cul1, and Rbx1 to confirm their presence; and GAPDH antibody to validate that equivalent amounts of protein were present in each sample. Whole-cell lysates were incubated with Flag antibody-conjugated agarose beads to immunoprecipitate (IP) Flag-tagged proteins and their interacting proteins. The resulting Western blot was probed with indicated antibodies to confirm recovery of the Flag-tagged proteins and assess for Skp1, Cul1, or Rbx1 coimmunoprecipitation. (C to E) Ankyrin repeat 1 (containing the putative NLS) and the F-box of Ank13 are dispensable for nuclear localization. Transfected HeLa cells expressing Flag-Ank13, Flag-Ank1349-490, or Flag-Ank13ΔF-box were either fixed, probed with Flag antibody, stained with DAPI, and examined by immunofluorescence microscopy (C) or lysed and resolved into cytoplasmic [C] and nuclear [N] fractions that were subjected to Western blot analyses using the indicated antibodies (D). (E) The nuclear densitometric value was divided by the sum of nuclear and cytoplasmic densitometric values for Flag-tagged proteins in panel D. The quotient was multiplied by 100 to yield the percentage of Flag-tagged protein in the nucleus. Data presented are the percentages (means ± the SD) of Flag-tagged proteins exhibiting nuclear localization from three separate experiments. One-way ANOVA with Tukey’s post hoc test was used to assess for significant differences among the conditions. n.s., not significant.

Fig 4: Expression characteristics of miR-21-3p and miR-769-3p from RNA and exome sequencing datasets of colon cancer from TCGA. (A) Spearman’s correlation analysis of miR-21-3p and miR-769-3p expression in colon cancer tumor tissues (n=425); (B) A somatic mutation of p53 gene presented in 415 patients. MiR-21-3p (left panel) and miR-769-3p (right panel) levels were significantly higher in the p53 mutant group (mut, n=67) than in the wild-type group (w/o mut, n=348). RPM, reads per million; **, P<0.01; ***, P<0.0001; (C, D) Expression of miR-21-3p (C) and miR-769-3p (D) were inversely correlated with mRNA levels of Smad7 and Skp1 in patients with colon cancer. Spearman’s rank correlation coefficients (rho) were calculated. RPM, reads per million; (E) Schematic diagram of positive feedback loop between tumor cells with mutant p53 and fibroblasts in the lung. Exosomes derived from p53 mutant cells activate stromal fibroblasts in lung tissues via miR-21-3p and miR-769-3p to form a premetastatic niche. Then, activated fibroblasts secrete transforming growth factor β (TGF-β) to induce epithelial-mesenchymal transition (EMT) of tumor cells, leading to a stronger invasive phenotype. Thus, circulating exosomes play central roles in the pulmonary metastatic feedback loop.

Fig 5: TCAP-FBXL21 and FBXL21-CULLIN1 Complex Formation Is Regulated by GSK-3ßGreen and blue colors denote Venus and DAPI signals.(A) TCAP and FBXL21 form a complex in the cytoplasm, and shGSK-3ß co-transfection inhibits complex formation. Right panel: the bar graph shows mean ± SEM from BiFC signal quantification; n = 3. One-way ANOVA shows that the relative fluorescence intensity is significantly statistically different between mock (scramble shRNA) and GSK-3ß shRNA. (***p < 0.001).(B) FBXL21-CULLIN1 complex formation was significantly inhibited by shGSK-3ß co-transfection. Right panel: the bar graph shows mean ± SEM of BiFC signal quantification; n = 3. One-way ANOVA shows a significant statistical difference between mock and GSK-3ß shRNA (****p < 0.0001).(C) FBXL21-SKP1 complex formation was not affected by shGSK-3ß co-transfection. Right panel: the bar graph shows mean ± SEM; n = 3. One-way ANOVA shows that the relative fluorescence intensity is statistically not different. Scale bar, 20 µm.(D) FBXL21T33A showed reduced Cullin1 complex formation compared with FBXL21. Right panel: the bar graph shows mean ± SEM; n = 3. One-way ANOVA shows a significant statistical difference between Fbxl21 WT and Fbxl21T33A (***p < 0.001) and Fbxl21T33A.GSK-3ß shRNA (****p < 0.0001). Scale bar, 20 µm.See also Figure S5.