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. 2017 Jan 4;114(3):E317–E326. doi: 10.1073/pnas.1614684114

Fig. 6.

Fig. 6.

BRAFV600E expression combined with Dusp5 deletion causes ERK hyperactivation and proliferative arrest. (A) Primary WT and Dusp5 KO MEFs were infected with empty Ad, Ad HA-HRASQ61L, or Ad HA-BRAFV600E alongside 0.3–3 pfu/nL of Ad expressing EGR1 promoter-driven DUSP5-Myc before stimulation with 20% (vol/vol) FBS. Plots show time courses comparing whole-cell p-ERK levels using HCM. Normalized population mean AFU values are shown ± SEM, n = 4. *P < 0.05, **P < 0.01 comparing KO vs. KO + 3.0 pfu/nL Ad DUSP5-Myc using two-way repeated-measures ANOVA and Bonferroni posttest. (B) Immortalized WT and Dusp5 KO MEFs were infected with retrovirus control, HRASQ61L, or BRAFV600E vectors for 2 wk before assessment of transformed foci using crystal violet staining. Representative plate scans are shown and plots show average focus formation per well ± SEM, n = 4. **P < 0.01 comparing control and oncogene-expressing cells using two-way repeated-measures ANOVA and Bonferroni posttest. (C) Primary WT and Dusp5 KO MEFs were infected with either empty Ad, Ad HA-HRASQ61L, or Ad HA-BRAFV600E and treated with increasing doses of PD0325901 MEK inhibitor for 24 h before a 2-h fluorescent EdU pulse label, immunostaining for p-ERK, and analysis using HCM. Plots show Nuc p-ERK levels and the percentage of EdU-positive cells ± SEM, n = 3. **P < 0.01 using two-way repeated-measures ANOVA and Bonferroni posttest comparing WT and KO. Representative images are shown beneath. (Scale bar: 200 μm.) (D) WT and Dusp5 KO primary MEFs were infected with Ad HA-BRAFV600E (Lower), Ad HA-HRASQ61L (Upper), and 0.3 pfu/nL Ad DUSP5-Myc and treated with 0.1 µM PD0325901 (PD) as indicated for 24 h before 2-h fluorescent EdU pulse-labeling of S-phase cells and counterstaining for p-ERK and HA-tag. The plots show HA intensity vs. Nuc p-ERK in single cells; contour lines show equal cell density; and the heat map reflects the percentage of S-phase–positive cells within HA and p-ERK bins. Data plotted are in raw AFU and population statistics, including mean p-ERK levels, p-ERK SD, and overall percentage of S-phase–positive cells are shown within individual plots.