Fig 2: Loss of AKAP8 protein abrogates the DNA damage effect seen in neighbouring cells.a Sanger sequencing trace of RKO cell line showing an isogenic AKAP8-/- clone with 20 nucleotide deletion (blue box) from wildtype. b Western blot validation showing loss of FBXW7 or AKAP8 protein in RKO cells. c Sanger sequencing trace of LS513 cell line showing an isogenic AKAP8-/- clone with 16 nucleotide deletion (blue box) from wildtype. d Western blot validation showing loss of FBXW7 or AKAP8 protein in LS513 cells. RKO wildtype cells were co-cultured with either single mutant FBXW7-/- cells or double mutant FBXW7-/-/AKAP8-/- cells. In RKO cells, ?H2AX (p = 0.0011; Fig. 7e, g) and 53BP1 (p < 0.0001; Fig. 7e, i) foci were decreased in double mutant cells compared with single mutant cells. Similarly, LS513 wildtype cells were co-cultured with either single mutant FBXW7-/- cells or double mutant FBXW7-/-/AKAP8-/- cells. In LS513 cells, ?H2AX (p = 0.0285; Fig. 7f, h) and 53BP1 (p = 0.0479; Fig. 7f, j) foci were decreased in double mutant cells compared with single mutant cells. Boxplots are presented as 10–90 percentiles with the median value represented by a line across the box, and an asterisk to denote the mean. Statistical significance was measured by unpaired t-test. All experiments were performed with N = 3 biological replicates.

Fig 3: DNA damage was increased in wildtype cells which were co-cultured with FBXW7-/- cells.a Schematic showing how cells were cultured on the Transwell insert. Cells are cultured on both sides of the insert and are therefore in close proximity with one another without cells being able to migrate across the membrane. b In RKO cells, ?H2AX foci per nuclei were significantly increased between wildtype and all other groups. Notably, ?H2AX foci were increased between WT and CC-WT cells. (WT vs. CC-WT, p = 0.0032; WT vs. CC-Mut, p < 0.0001; WT vs. CC-Mut-, p < 0.0001; CC-Mut vs. Mut, p = 0.0154.) This same effect was observed when staining for 53BP1 foci. (WT vs. CC-WT, p < 0.0001; WT vs. CC-Mut, p < 0.0001; WT vs. Mut, p < 0.0001; CC-Mut vs. Mut, ns.) c In LS513 cells, ?H2AX foci per nuclei were significantly increased between wildtype and all other groups. As in RKO cells, ?H2AX was increased between WT and CC-WT cells (WT vs. CC-WT, p = 0.0404; WT vs. CC-Mut, p = 0.0189; WT vs. Mut, p = 0.0029; CC-Mut vs. Mut, ns). A similar effect was observed when staining for 53BP1 foci (WT vs. CC-WT, p = 0.0022; WT vs. CC-Mut, p = 0.0004; WT vs. Mut, p = 0.0052; CC-Mut vs Mut, ns). Immunofluorescent images of nuclei with ?H2AX foci in RKO cells (d) and LS513 cells (f). Immunofluorescent images of nuclei with 53BP1 foci in RKO cells (e) and LS513 cells (g). All scale bars represent 5 µm. Boxplots are presented as 10–90 percentiles with the median value represented by a line across the box, and an asterisk to denote the mean. Statistical significance was measured by unpaired t-test. All experiments were performed with N = 3 biological replicates.

Fig 4: Characterisation of wildtype and FBXW7-/- cells.a Sanger sequencing trace of RKO cell line showing an isogenic FBXW7-/- clone with 22 nucleotide deletion (blue box) from wildtype. b Sanger sequencing trace of LS513 cell line showing an isogenic FBXW7-/- clone with 1 nucleotide insertion (blue box) from wildtype. c Western blot validation showing loss of FBXW7 protein in RKO cells. d Western blot validation showing loss of FBXW7 protein in LS513 cells. e Live-cell proliferation analysis using IncuCyte imaging system showed increased proliferation of wildtype cells (green) relative to FBXW7-/- cells (red). (p < 0.0001). f EdU incorporation assay showed increased proliferation occurring in wildtype cells relative to FBXW7-/- cells (p < 0.0001). g RT-PCR showed increased MKI67 (p = 0.0002) and PCNA (p = 0.0003) in wildtype relative to FBXW7-/- cells. h No statistically significant differences in both early and late apoptotic markers (blue: Annexin V and 7-AAD negative; violet: Annexin V positive, 7-AAD negative; orange: Annexin V and 7-AAD positive). i No difference in cleaved caspase 3 levels to suggest increased apoptosis accounting for the observed difference in cellular proliferation (N = 3). The values in this figure are mean ± sd, and statistical significance was measured by unpaired t-test. All experiments were performed with N = 3 biological replicates.

Fig 5: RNAseq results between wildtype and FBXW7-/- cells.a Heatmap showing the top 50 differentially expressed genes between wildtype and FBXW7-/- cells. b Transcript levels for genes which act as direct substrates for FBXW7. All differences were non-significant except for MYC (p = 0.0008) which was unexpectedly decreased in an FBXW7 knockout, possibly due to negative feedback homeostatic regulation. c Transcript differences in metalloproteinases which were observed to be upregulated in FBXW7-/- cells. All were significant with p < 0.0001. The values in this figure are mean ± sd, and statistical significance was measured by unpaired t-test. Biological triplicates, N = 3, were submitted for RNAseq.