Fig 2: PLO induces phosphorylation of MAPK. A) Cytolysis of endometrial stromal cells treated for 1, 2, or 4 hours with the indicated concentration of PLO, as determined by MTT assay. B) Western blotting analysis of total (t-) and phosphorylated (p-) ERK1/2, p38, and JNK in endometrial stromal cells treated with 50 HU PLO or 1 µg/ml LPS for the indicated times, with a-tubulin (TUBA) used as a loading control (CTRL). C) Western blotting analysis of phosphorylation of ERK in endometrial stromal cells treated with 50 HU PLO or 50 HU dsPLO for the indicated times, with a-tubulin used as a loading control. Data in graphs are presented as the mean (SD) of 3 independent experiments, and data were analyzed using ANOVA and Dunnett’s post hoc test. Values differ from control: *P < 0.05; **P < 0.01; ***P < 0.001. D) Western blotting analysis of phosphorylation of ERK in endometrial stromal cells treated with 50 HU PLO, after 1 hour preincubation with medium alone or medium containing a-PLO antibody, with a-tubulin used as a loading control.

Fig 3: Compensatory functions of BRAF and CRAF for cell proliferation and tumour growth in NRASQ61K-induced murine melanoma.(a,e) A melanoma from an untreated Braff/f;Craf+/+;Tyr::NRASQ61K/o;Ink4a+/-;Tyr::CreERT2/o or Braf+/+;Craff/f;Tyr::NRASQ61K/o;Ink4a+/-;Tyr::CreERT2/o mouse (a,e, respectively) was cut into small pieces and subcutaneously grafted into two groups of nude mice and experimented as in Fig. 2a. These experiments required 48 Swiss Nu/Nu females (6-week-old) for each primary tumour from a 5-month-old female and a 1-year-old male on a SV129/C57Bl6 mixed genetic background, respectively. (b,f) Western blot analysis for BRAF and CRAF expression at the end of treatment by tamoxifen or vehicle in three individual and representative tumours from a,e, respectively. ß-actin is used as a loading control. (c,g) Growth curve analysis of melanoma cell culture established from an untreated Braff/f;Craf+/+;Tyr::NRASQ61K/o;Ink4a+/-;Tyr::CreERT2/o or Braf+/+;Craff/f;Tyr::NRASQ61K/o;Ink4a+/-;Tyr::CreERT2/o primary mouse tumour (c,g, respectively) in response to 4OHT or DMSO for 9 days as in Fig. 2c. (d,h) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels in protein lysates from culture in c,g respectively, as in Fig. 2b. All data are represented as mean±s.d.

Fig 4: Effect of MEK1/2 inhibition on NIS transcriptional expression in thyroid cells expressing NRAS and BRAF gain-of-function mutants. (A) PCCL3 cells transduced as indicated were serum-starved for 24 h followed by stimulation with TSH (1 mU/mL for 48 h) in the presence or absence of either AZD6244 (10 µM for 48 h) or CH5126766 (10 µM for 48 h). NIS mRNA levels were quantified by RT-qPCR and plotted as fold differences relative to mock (CFP)-transduced cells, without treatment with the MEK inhibitors [Ctrl (empty)]. Values are means ± SEM of five independent assays. Comparisons were made using a two-tailed Student’s t-tests between AZD6244-treated or CH5126766-treated versus untreated setting (ns—not significant; ** p = 0.01; *** p = 0.001). (B) NRAS and BRAF expression was assessed by western blot using anti-GFP primary antibody. MAPK activation status was assessed by monitoring phosphorylated ERK 1/2 vs. total ERK 1/2 levels by western blot [quantified below the respective panel (means ± SEM of three independent assays)], using anti-phospho ERK 1/2 and anti-ERK 1/2 primary antibodies, respectively. Alpha-tubulin was used as loading control (see Figure S3 for uncropped WB).

Fig 5: Stepwise regression of Erk activity in control embryos. (a) A line of interest for kymograph analysis in a control embryo (3-color heat map). Scale bar, 50 µm. (b) Kymograph of the control embryo. Horizontal and vertical axes indicate the length from the anterior end of the PSM (µm) and the progression of time (min), respectively. Scale bar, 50 µm. Signal intensities are shown by the heat map. The original movie is provided as Movie 3. (c) A contour corresponding to the Erk activity border was extracted from the kymograph (panel b). Arrowheads indicate the change-points of the curve and correspond to the time of the stepwise shifts of the Erk activity border. Time-dependent positional changes could be seen in the control embryo. (d) Distribution of time interval among the stepwise shifts of Erk activity. The distribution was generated from control embryos (n = 5). The black line indicates the distribution of time intervals for all stepwise shifts. Red, blue, green, and yellow shaded regions correspond to the distribution of time intervals between the indicated and the next stepwise shifts, or the subsequent two, three, or more than four consecutive stepwise shifts, respectively. Red, blue, green and yellow dots indicate the data points.