Fig 2: Fbxw7 deletion before osteogenic induction suppresses differentiation of MSCs to osteoblasts.a Diagram showing an experimental setup for ex vivo deletion of Fbxw7. Isolation of MSCs from the bone marrow of UbcCreERT2; Fbxw7f/f mice (1) was followed by 4OHT-induced deletion of Fbxw7 (2), osteogenic induction (3) and analysis of osteoblast differentiation (4). b–e Osteoblast differentiation of UbcCreERT2; Fbxw7f/f-derived MSCs treated with mock or 4OHT before osteogenic induction (n = 3 different mice). Differentiation was measured at 14, 21, and 28 days after osteogenic induction via Alizarin Red S staining (b) and mRNA levels of osteoblast differentiation markers Runx2, Osterix (Osx), Osteocalcin (Ocn), Osteopontin (Opnt) and Alkaline phosphatase (Alp) at 14 (c), 21 (d), and 28 days (e) after osteogenic induction. mRNA levels of Fbxw7 were analyzed to confirm deletion of the gene. Images were taken from 96-well plates. Well diameter: 5 mm (b). Data are presented as means ± SEM. Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Identification of the T244 degron.(A) HEK293 cells were cotransfected with HA-Myc and FLAG-Fbw7, and lysates were immunoprecipitated (IP) with FLAG antibody and blotted with HA antibody. F-box deletion (∆F) uncouples binding from turnover. (B) Alignment of the T58 and T244 degrons. (C and D) Assays as in (A). To isolate the T244 degron, all mutants were made in a T58A mutant background in (D). S252 was tested for its potential role as a priming site for degron phosphorylations. (E) Quantitative peptide competition analysis of Myc peptides with a cyclin E pT380/pS384 degron peptide for Fbw7 binding as measured in arbitrary fluorescent units (AFU). (F) Myc or mutants were immunoprecipitated from transfected HEK293 cell lysates, and washed beads were subjected to in vitro ubiquitylation. Fbw7 was omitted in lane 4 (−Fbw7) as negative control. Reactions were analyzed by Western blotting. (G) HeLa and U2OS cell lines were treated with proteasome inhibitor Velcade for 4 hours (lanes 2 and 4), and lysates were immunoprecipitated against Myc and blotted as indicated. While Myc levels increase upon proteasome inhibition, pT244 appears to be increased relative to total Myc levels. (H) Isogenic DLD1 WT cells or their homozygously mutated cell lines (T244A or Fbw7 deletion) were treated with MLN4924 (right lanes only), and lysates were immunoprecipitated against Myc and blotted as indicated. Asterisk marks an antibody cross-reaction of the IP antibody heavy chain. (I) DLD1 cells or isogenic derivatives homozygously deleted for Fbw7 or T244A Myc mutated were treated with cycloheximide (CHX) and blotted for Myc steady-state abundance. Proliferating cell nuclear antigen (PCNA) serves as loading control. Bands were quantitated and plotted on the right.

Fig 4: The effect of GUTK on cell cycle re-entry was diminished after FBXW7 knockdown. LNCaP cells following 5-day serum withdrawal and PC-3 cells at the day of contact inhibition were transfected with 50 nM siRNA duplexes targeting scramble or FBXW7 (#1 and #2) for 48 h (LNCaP) and 72 h (PC-3). FBXW7 α and β isoforms and c-MYC protein in quiescent LNCaP (a) and PC-3 (c) cells were analyzed by immunoblotting. GAPDH served as a loading control. The mRNA levels of FBXW7 were detected by RT-qPCR in quiescent LNCaP (b) and PC-3 (d) cells. TBP was used for normalization. After FBXW7 knockdown, c-MYC protein levels in LNCaP (e) and PC-3 (f) cells, which were induced for cell cycle re-entry with or without GUTK at GI75, were analyzed by immunoblotting. GAPDH served as a loading control. The effect of GUTK on DNA content in FBXW7-knockdown LNCaP (g) and PC-3 (h) cells after release from quiescence for 24–72 h was determined by SYBR green assay. The growth inhibition was calculated using the equation descried under SYBR Green assay in Materials and Methods. The data are expressed as the mean±S.D. of triplicate assays, *P<0.05, **P<0.01, ***P<0.001, compared with the corresponding negative control (NC)

Fig 5: TaPin1 changes c-Jun stability through the regulation of FBW7a. TaPin1 interacts with endogenous mouse FBW7. Protein extracts from NIH3T3 cells stably expressing HA-Flag-TaPin1 or HA-Flag-Control “Con” were used for HA immunoprecipitation, followed by immunoblot analysis using FBW7 and HA antibodies.b. TaPin1 interacts with endogenous bovine FBW7 protein. Parasite-infected TBL3 and TpMD409 cell lysates were incubated with indicated GST-coupled beads, followed by immunoblot analysis using a FBW7 antibody.c/d Analysis of indicated bovine gene expression by qPCR in TBL3 cells following Buparvaquone (Bup) or Juglone (Jug) treatment with or without TaPin1 WT or Mutant A53P. ß-actin and H2A mRNAs were used for normalization (data represent 3 independent experiments - average ± sd, n=3).e. siPin1 does not affect FBW7a or c-Jun protein levels. TBL3 were transiently transfected by siControl “siCon” or si-bovine-Pin1. Indicated protein levels were detected by Western blot analysis using specific antibodies. Actin was used as loading control.f. Inhibition of TaPin1 by Buparvaquone/Juglone increased c-Jun ubiquitination and decreased FBW7 ubiquitination. Parasite-infected TBL3 cells (+/- Buparvaquone (Bup) or Juglone (Jug) - “Con” indicates Control) were incubated with MG132, followed by immunoprecipitation of endogenous c-Jun or FBW7 and immunoblot analysis with the indicated antibodies.g. The effect of Buparvaquone on c-Jun ubiquitination was rescued by overexpression of TaPin1 Mutant A53P. Parasite-infected TBL3 cells (+/- Buparvaquone (Bup) or Juglone (Jug) - “Con” indicates Control) were transfected with TaPin1 WT, TaPin1 Mutant A53P or empty vector “Con” and then treated with MG132, followed by immunoprecipitation of endogenous c-Jun and immunoblot analysis with the indicated antibodies.h. Measure of c-Myc, KLF5 and activated Notch 1 protein levels in TBL3 upon Buparvaquone (Bup) treatment. “Con” indicates Control. Tubulin was used as loading control.i. Measure of c-Myc, KLF5 and activated Notch 1 protein levels in TBL3 upon FBW7a ectopic expression. “Con” indicates Control = transfection with the appropriate empty vector. Tubulin was used as loading control.All the results in a, b, e, f, g, h and i are representatives of 3 independent experiments.