Fig 2: Enhanced soft agar growth with combined Dusp4 loss and Trp53 (p53) deletion. A Crystal violet 2D culture assay of PMECs transduced with CRISPR/Cas9 constructs targeting Trp53 (exon 7 or exon 5) or scramble control (Parent) and selected with 10uM nutlin-3a for 1 week, or DMSO control. Trp53 CRISPR transductants demonstrate insensitivity to MDM2 inhibition, verifying loss of p53 function. B Soft agar colony formation assay for Dusp4FLOX or Dusp4NULL PMECs transduced with control sgRNA or Trp53 sgRNA. C Western blot analysis of derivative PMECs

Fig 3: Combined Dusp4 and p53 loss induces multinucleation and eventual escape from replication stress. A Representative images of polyploidy/multinucleation in Dusp4 NULL cells. B Quantification of nuclear content in primary mammary epithelial-derived cell lines with various genetic alterations. C Representative images of Dusp4NULL cells stained with DAPI to visualize nuclear DNA content. D Cells were labeled with EdU and counterstained with DAPI and analyzed by flow cytometry to analyze DNA synthesis. Representative flow cytometry gating is shown. E S phase analysis by EdU *Tukey’s post hoc p < 0.05** p < 0.001. F G2 phase analysis demonstrating increased G2 phase in Dusp4NULL p53Δ and DPM cells. G Whole-cell lysates were immunoblotted for replication stress marker pCHK1

Fig 4: Changes in O-GlcNAcylation amplify ERK 1/2 signaling. Arrows in magenta indicate long term OGA inhibition via TMG treatment for at least 2 weeks. Green arrows indicate OGT KD via shRNA 605. Blue arrows indicate OGT KD via shRNA 606. Purple arrows indicate OGA KD via shRNA 040. Orange arrows indicate OGA KD via shRNA 877. OGA inhibition via long term TMG treatment, OGT KD and OGA KD, all increase the amplitude of ERK 1/2 phosphorylation. TMG treatment increases ERK 1/2 phosphorylation by increasing the amplitude of MEK 1/2 phosphorylation. The levels of DUSP4 does not change with TMG treatment. OGT KD 606 also increases ERK 1/2 phosphorylation by increasing the amplitude of MEK 1/2 phosphorylation. The levels of DUSP4 decrease with OGT KD 606. OGT KD with shRNA 605 follows a similar trend as OGT KD with shRNA 606 where the levels of DUSP4 decreases. But the increase in MEK 1/2 phosphorylation by OGT KD 605 is not significant. OGA KD with both shRNA 040 and 877 increase ERK 1/2 phosphorylation but increase in the level of MEK 1/2 phosphorylation was not significant. DUSP4 decreases significantly with OGA KD 877 and DUSP4 levels do not change with OGA KD 040. Long term OGA inhibition also increased APP protein levels, correlating with increased ERK phosphorylation.

Fig 5: DUSP4 silencing–induced ERK overactivation plays an essential role in causing toxic effects of MITF-proficient melanoma cells independently of the oncodriver mutation.(A, B) High-MITF–expressing cells (SKMEL28, SKMEL30) and low-MITF–expressing cells (A375 and SKMEL2) were transfected either with single siRNA against nontargeting control, DUSP4, and DUSP6 or co-transfected with siRNAs against DUSP4 and DUSP6 (siDUSP4+6) or DUSP4 and DUSP10 (DUSP4+10). (A, B) After 48 h, cell lysates were analyzed by Western blot (A), and DUSP10 mRNA levels were measured by RT–qPCR (B). Band intensities (A) were analyzed by ImageJ software, and the P-ERK/GAPDH and MITF/GAPDH ratios are indicated in the histogram. Data represent mean ± SEM of three independent experiments. Statistical significance was calculated against siNT for each different cell line. (A, C) Cells were treated as in (A), and cell growth was analyzed by measuring cell confluence over time. Graphs show cell growth curves normalized against time 0. Data are mean ± SEM, n = 2.