Fig 2: Association of TRAF7 with SCRIB(A) Western blot analysis of SCRIB protein expression and its co-immunoprecipitation (co-IP) with TRAF7 using HEK293 whole-cell lysates and indicated antibodies. BAP (bacterial alkaline phosphatase), COP1, TRAF7, or TRAF7N520S were overexpressed as FLAG-fusion proteins, and co-IP was done with anti-FLAG antibody. Co-IP lanes show amount of protein in 10 times lysate input volume.(B) Schematic structure of TRAF7 protein. S/T-rich (serine/threonine-rich), RF (ring finger), ZF (Zinc finger), CC (coiled coil), and WD40 domains are depicted by different colors. Amino acids numbers (aa #) above the diagram correspond to border amino acids in truncated FLAG-tagged TRAF7 proteins used in this study.(C) Western blot analysis of MEKK3, MEK5, and SCRIB protein expression and their co-immunoprecipitation (co-IP) with overexpressed truncated FLAG-TRAF7 proteins using HEK293 whole-cell lysates and indicated antibodies. Co-IP was done with anti-FLAG antibody. Anti-α-Tubulin antibody was used as the loading control. Co-IP lanes show amount of protein in 10 times lysate input volume. Blots shown in panels (A) and (C) are representative of at least four independent experiments.

Fig 3: MEKK3‐MEK5‐dependent ERK5 phosphorylation is responsible for KLF4 upregulation Left panel: WB analysis of pERK5, ERK5, KLF4, and MEKK3 in WT and CCM1 KO ECs treated with either two different siRNAs against Mekk3 (siMEKK3 #1 and siMEKK3 #2) or a control sequence (siCTRL). Both pERK5/ERK5 ratio and KLF4 amount normalized over vinculin were quantified by densitometry scan. WB results are representative of three independent observations. Right panel: qRT–PCR of Klf4 in WT and CCM1 KO ECs treated with two different siRNAs directed to Mekk3 (siMEKK3 #1 and siMEKK3 #2) or a control sequence (siCTRL). qRT–PCR results are shown as mean ± SD (n = 3), and fold changes are relative to siCTRL‐treated WT ECs. A two‐tailed unpaired t‐test was performed. ***P = 0.0002, **P = 0.002, ****P = 2.5E‐05, #### P = 3.1E‐05, ### P = 0.0007. WB analysis of pERK5, ERK5, KLF4, and MEK5 (left panel) and qRT–PCR of Klf4 (right panel) in WT and CCM1 KO ECs treated with a siRNA against MEK5 or a control sequence (siCTRL) quantified as in (A). **P = 0.0035, ****P < 0.00001, #### P = 3.5E‐05. WB analysis of pERK5, ERK5, and KLF4 (upper panel) and qRT–PCR of Klf4 (lower panel) in WT and CCM1 KO ECs treated with BIX‐02189 or vehicle for 48 h quantified as in (A). Fold changes are relative to vehicle‐treated WT ECs. A two‐tailed unpaired t‐test was performed. ***P = 0.0002, ****P = 2.1E‐05.Schematic model of KLF4 activity and regulation during CCM pathogenesis.Source data are available online for this figure.

Fig 4: miR-3140-3p suppressed the growth of BETi-acquired resistant NB cells by concurrently targeting BRD4 and the MAP3K3-ERK1/2 pathway(A) Western blotting of the indicated proteins in IMR-32 and IMR-32-JQ1R cells transfected with 10 nmol/L miR-NC or miR-3140-3p. (B) Cell growth assay (upper) and phase-contrast images (lower) of IMR-32 and IMR-32-JQ1R cells 72 h after transfection with 10 nmol/L miR-NC or miR-3140-3p. The cell growth rate was assessed by WST-8 assay using a relative ratio compared with miR-NC-transfected cells. Bar, SD for triplicate experiments. ∗ P < 0.05. (C) Western blotting for MAP3K3, ERK1/2, and p-ERK1/2 in IMR-32 and IMR-32-JQ1R cells transfected with 20 nmol/L si-NC or si-MAP3K3. (D) Dose-response curve for cell viability using the WST-8 assay in IMR-32-JQ1R cells after treatment with different concentrations (0, 0.031, 0.063, 0.13, 0.25, 0.50, 1.0, or 2.0 µmol/L) of JQ1 for 48 h combined with transfection with si-NC or si-MAP3K3. The cell growth rate was assessed using a relative ratio compared with that of DMSO-treated cells. Bar, SD for triplicate experiments. ∗ P < 0.05. (E) Western blotting of the indicated proteins in control cells (IMR-32-EV) and MAP3K3-overexpressing stable clone (IMR-32-MAP3K3). Dose-response curve for cell viability using the WST-8 assay in IMR-32-EV and IMR-32-MAP3K3 cells after treatment with different concentrations (0, 0.031, 0.063, 0.13, 0.25, 0.50, 1.0, or 2.0 µmol/L) of JQ1 for 48 h. The cell growth rate was assessed using a relative ratio compared with that of DMSO-treated cells. Bar, SD for triplicate experiments. ∗ P < 0.05. (F) Cycloheximide (CHX) chase assay of IMR-32-EV and IMR-32-MAP3K3 cells, as described in Figure 3A. Western blotting (upper) and the quantification of MYCN at each time point (lower). The values were normalized against β-actin for each sample. Bar, SD for 3 independent experiments. (G) Ubiquitinated MYCN in IMR-32-EV and IMR-32-MAP3K3 cells. Amounts of polyubiquitin were measured as described in Figure 3B. (H) Luciferase reporter assays. IMR-32 cells were cotransfected with pmirGLO dual-luciferase vectors containing the wild-type (WT) 3’UTRs of MAP3K3 or mutant variants (Mt) of MAP3K3 and miR-NC or miR-3140. Upper, a putative binding site of miR-3140 within the 3’UTR of MAP3K3 and mutant sequences. Lower, the results of the luciferase assay; ∗P < 0.05.

Fig 5: Representative follow-up MRI scans of MAP3K3 mutation and CCM gene mutation lesions before surgical resection. (A) A patient who harbored a MAP3K3 mutation lesion remained stable based on follow-up MRI scans at 13 and 40 months. (B) A patient who harbored a CCM1 mutation lesion exhibited recurrent overt hemorrhage based on follow-up MRI scans at 3 and 31 months.