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: MEK1 knockdown suppresses liver CSC self-renewal and tumorgenetic capacity(A) Western blot analysis MEK1 and the substrate ERK1/2 expression in Huh7- and PLC/PRF/5-NanogPos cells which depleted MEK1 with two individual lentiviruses for 48 hours. (B) Effect of MEK1 knockdown on cellular growth rates of Huh7- and PLC/PRF/5- NanogPos cells. CCK8 assay was performed after transfection with indicated times. Cell lysates were obtained from cells transiently transfected with either MEK1 shRNA or negative control shRNA. (C) Huh7- and PLC/PRF/5- NanogPos cells which transfected with MEK1 shRNA or negative control shRNA cultured under non-adhesive culture system for 7 days. (D) Huh7- and PLC/PRF/5-NanogPos cells were transduced with lentiviruses expressing the indicated shRNA. Cells were grown for 14 days and stained with crystal violet. (E) Western blot analysis of stemness-related proteins in MEK1-depleted cells, relative to control.

Fig 4: TBMS1 induces hyperactivation of MEK1/2-ERK1/2 cascade in melanoma cells. (A) Western blot was performed to detect the expression levels of p-ERK1/2, ERK1/2, p-MEK1/2, and MEK1/2 in melanoma cells treated with TBMS1 for 48 h. (B) Western blot results showed the expression level of p-ERK1/2, ERK1/2, p-MEK1/2, and MEK1/2 and in melanoma cells treated with TBMS1 in different time for 0, 6, 12, 24, and 48 h. (C) Growth curve of MV3 and A375 cells treated with TBMS1 and 1.25 µM SL327. DMSO was used as control. (D) BrdU-positive MV3 and A375 cells after treated with TBMS1 and 1.25 µM SL327 for 48 h. DMSO was used as control. Quantifications of BrdU-positive MV3 and A375 cells were calculated. (E) Clonogenicity of MV3 and A375 cells treated with TBMS1 and 1.25 µM SL327. The formation of clones after 10 days culture. On the right is the quantitative figure of clonogenic assay results. A two-tailed unpaired Student’s t-test was carried out. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: Src phosphorylates and enhances the activity of BCKDK ex vivo.a Src phosphorylated BCKDK in a dose-dependent manner under EGF stimulation (EGF: 80 ng/mL, 15 min). b Src promoted the phosphorylation of BCKDK in HEK293T cells (left panel). Mutation in BCKDK at Y246 significantly downregulated the phosphorylation effect of Src on BCKDK (right panel). (EGF: 80 ng/mL, 15 min). c EGF induced time-dependent phosphorylation of BCKDK in HCT116, LoVo, and SW620 cells. (EGF: 80 ng/mL. 0, 5, 10, and 15 min). d HT29 and SW620 cells were treated with dasatinib at the indicated concentrations (0, 10, 20, and 50 nM). (EGF: 80 ng/mL, 15 min). e Src was knocked down in HT29 and SW620 cells by nontargeting (shMOCK) shRNA or Src shRNAs (#1-#7). f The effects of Src knockdown on the phosphorylation of BCKDK and its downstream targets (MEK1/2 and BCKDHA) were detected by western blot. (EGF: 80 ng/mL, 15 min). g Expression of p-BCKDK (Y246), p-MEK1/2 (S221), and p-BCKDHA (S293) was detected in Src+/+ and Src-/- cells (EGF: 20 ng/mL, 30 min).