Fig 2: FBW7 lysines 404 and 412 are autoubiquitylated and required for TRIP12 interaction.a Schematic of in vitro GST-FBW7 ubiquitylation and mass spectrometry (MS) workflow. The cartoon shows the location of the identified ubiquitylated lysines (K) on FBW7. b Schematic of FBW7 deletion mutants showing ubiquitylation sites identified from experiment in a. c Western blots for MYC-FBW7 deletion mutants. GFP was used as a transfection control. d Western blots of the indicated FLAG-FBW7 wildtype, single, and double mutant proteins overexpressed in HEK293T cells. e Western blots showing the stability of the indicated FLAG-FBW7 proteins in HEK293T cells treated with 100 µg/ml cycloheximide (CHX) for the indicated time. GFP plasmid was used as transfection control and subsequently GFP and Actin blots were used as loading controls in c–e. f Western blots of ubiquitylated FLAG-FBW7 wildtype and K404/K412 mutant in HEK293T cells. g Western blots showing interaction of FLAG-FBW7 wildtype and K404/412 mutant with GFP-TRIP12 in HCT116 cells. All blots are representative of at least three independent experiments. See also Supplementary Fig. 5. Source data are provided as a Source Data file.

Fig 3: TRIP12 regulates chemotherapy resistance via MCL1 and FBW7.a Western blots showing levels of the indicated FBW7 substrates in HEK293T cells treated with a control siRNA or two independent siRNAs targeting TRIP12. b Western blots for TRIP12 and MCL1 in cells targeted with CRISPR/Cas9 SAM TRIP12 sgRNAs or an empty vector control. Tubulin used as a loading control. c Western blots for MCL1 from HEK293T cells treated with control and TRIP12 siRNA ± MG132. Tubulin used as a loading control. d Western blot for the indicated proteins in HEK293T cells treated with the indicated siRNAs. Blots in a–d are representative of at least three independent experiments. e Cell proliferation of HCT116 cells of indicated genotypes, as judged by cell counting on indicated days. Graph shows mean ± SD of three independent experiments. f Viability of HCT116 cells of the indicated genotypes as judged by CellTiter-Blue® viability assay in the presence or absence of 100 nM Taxol and shown as % viability relative to the untreated control. Bar graphs represent Mean ± SD of three independent experiments, **p = 0.004 and ***p = 0.0001. g Viability of colorectal cancer cell lines with the indicated genotypes, treated with 100 nM Taxol for 72 h as judged by CellTiter-Blue® assay and shown as % viability relative to the control. Bar graphs represent mean ± SD of three independent experiments, **p = 0.01 and *p = 0.04, n.s., not significant. p-values calculated by two-tailed type two Student’s t-test. See also Supplementary Fig. 3. Source data are provided as a Source Data file.

Fig 4: TRIP12 preferentially interacts with ubiquitylated FBW7.a Co-IP confirming endogenous FBW7 interaction with TRIP12 and vice versa in HEK293T cells, black star (*) represents a nonspecific band of unknown identity. b Schematic for experiments in c and d. c In vitro 35S-FBW7a ubiquitylation experiment in the presence of the indicated reagents. d In vitro deubiquitylation of 35S-FBW7a by USP2. e Schematic of in vitro translated 35S-FBW7 pulldown with MYC-TRIP12 from HEK293T cells. f Autoradiograph showing MYC-TRIP12 interaction with ubiquitylated FBW7. g Western blots showing the interaction of MYC-FBW7 with FLAG-TRIP12 in HCT116 cells pre-treated for 24 h with either NAE1 inhibitor (MLN4924) or DMSO as control. h Schematic of GFP-TRIP12 deletion mutants. i Western blots showing the interaction of MYC-FBW7 with GFP-TRIP12, GFP-TRIP12?HECT, GFP-TRIP12?HECT/ ?WWE mutants in HCT116 cells, black star (*) represents a nonspecific band of unknown identity. All blots are representative of at least three independent experiments. See also Supplementary Fig. 4. Source data are provided as a Source Data file.

Fig 5: Simultaneous p53 deletion abrogates cell senescence induced by FBXW7 deletion.A549 and MCF7 cells with deletion of the indicated genes using CRISPR/Cas9 technology were subjected to SA-β-Gal staining, followed by microscopy (A left) or IB with the indicated antibodies (B), or cell counting kit (CCK)-8-based cell proliferation assay (C). The percentage of SA-β-Gal-positive cells in the total number of cells was determined (A right). Scale bars represent 50 μm. Data are presented as the mean ± SD. *p < 0.05, ***p < 0.001. Cell proliferation is expressed as a fold-change compared to day 1. Data are presented as the mean ± standard error of the mean (SEM) from three independent experiments. n = 3; **p < 0.01, ***p < 0.001.