Fig 2: MEK1 inhibitor decreases liver CSCs proliferation ability in vitro(A) Huh7-NanogPos and PLC/PRF/5-NanogPos cells under different U0126 concentrations treatment as indicated (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) were seeded (1 × 103) and cultured for another 6 days before analyzed with CCK8. (B) Huh7-NanogPos and PLC/PRF/5-NanogPos cells were cultured with or without 5 μM U0126 for 48 hours. Cells were harvested for immunofluorescence (IF) analysis by anti-Ki67 antibodies. Scale bar, 10 μm. (C) Cell cycle profiles of 5 μM U0126 treated or DMSO treated (negative control) Huh7- and PLC/PRF/5-NanogPos cells followed by treatment with Sodium butyrate. Percentage in each histogram indicates the portion of cells remaining in each cell cycle phase.

Fig 3: BCKDK directly phosphorylates MEK1 at ser221. (a) BCKDK binds with MEK in HEK 293 T cells after transient transfection as indicated. pCMV-Flag-BCKDK or pCMV-Myc-MEK1 construct was trasfected or co-transfected into HEK 293T cells, immunoprecipitated with an anti-Flag or anti-Myc antibody, and then probed with Flag or anti-Myc antibody, respectively. (b) BCKDK binds with MEK in HCT116 cells. Endogenous BCKDK was immunoprecipitated from HCT116 cells and then probed with anti-MEK antibody. (c) BCKDK phosphorylates MEK1 in vitro. The samples in A were subjected to immunoprecipitation using the mouse source Flag antibody, and then a kinase assay was performed with His-MEK1(residues 62-393) as substrate, and corresponding rabbit source antibodies were used in Western blot analyses. (d) BCKDK promotes phosphorylation of endogenous MEK in HEK 293 T cells in a dose-dependent manner after EGF treatment for 15 min. Increasing amounts of Flag-BCKDK were transiently transfected into HEK 293 T cells, and the level of phosphorylation of MEKs was detected by Western blot. All Western blot data are representatives of results from triplicate experiments.

Fig 4: The MAPK pathway is involved in CCL28-mediated tumor metastasis. (A) Western blot analysis of MMP2, MMP9, VEGF and β-catenin expression in MDA-MB-231HM/vector (231HM/vector) and MDA-MB-231HM/CCL28 (231HM/CCL28) cells. (B) β-catenin protein expression was detected by immunohistochemical analysis in xenografts of 231HM/vector and 231HM/CCL28 tumor-bearing mice. (C) Association of CCL28 with the MAPK signaling pathway. Overexpression of CCL28 increased the level of phosphorylated ERK1/2, MEK1/2, and JNK1/2 as determined by western blot analysis. (D) Immunohistochemical examination of pERK protein expression in xenografts of 231HM/vector and 231HM/CCL28 tumor-bearing mice. MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor.

Fig 5: Mek inhibitor treatment reduced the levels of upregulated pMek, pErk, interstitial infiltrates, tubular dilations, and fibrosis in TNS1-KO kidneys.TNS1-WT or KO male C57 mice (N = 4, each group) at 3-month-old were treated with trametinib (1 mg/kg) or vehicle for 14 days. Kidneys were harvested 4 days post-treatment and processed for H&E, IHC staining using pMek1/2(pS221), Mek1/2, or Erk(pT202pY204) antibodies, and Sirius Red staining for collagen. Interstitial infiltration and IHC signals were quantified for statistical analysis shown on the left panel. Note that levels of pMek and pErk, and signs of interstitial infiltrates, fibrosis and dilated tubules were significantly reduced in TNS1-KO kidneys treated with trametinib. Arrows show dilated tubules. Arrowhead indicates interstitial infiltrates. Scale bars = 50 μm in Sirius Red, or 100 μm in others. P-values were calculated using two sample t-test. ***P < 0.0001.