Fig 2: EVI1 downregulates miR-449a in NPC cells. (A) Expression of miR-449a in C666-1 cells transfected with EVI1 siRNA or pEFzeo-EVI1 plasmid. (B) Expression levels of miR-449a in ICG-001-treated EBV-positive (C666-1 and C17) and EBV–negative (Hone-1) NPC cells. (C) Effect of miR-96 on the expression of miR-449a in C666-1 cells. At least three individual experiments were performed. * p < 0.05, ** p < 0.01 compared with the corresponding control.

Fig 3: ETV6-MECOM binds to−1116TTAAAA−1111in the E-cadherin promoter. (A) ETV6-MECOM inhibited the transactivation of E-cadherin promoter. (B) A diagram showing pGL3-E-cadherin truncations (−2100, −1200, −997, −672, −488 and −236). (C) Luciferase activity assay results showing that ETV6-MECOM regulated E-cadherin at −1200 to −997. (D) Bioinformatic analysis predicting putative binding sequences in the E-cadherin promoter. (E) Luciferase activity assay results showing the putative sequence 5′-AAGACTAGATTAAAA-3′ as the regulatory region of the ETV6-MECOM oncoprotein. (F) A diagram showing pGL3-E-cadherin-1200 mutations (MUT-1123 to −1117 and MUT-1110 to −1108) and deletion (Δ-1116 to −1111). (G) Luciferase assay results showing that only the Δ-1116 to −1111 plasmid inactivated the E-cadherin promoter. (H) A diagram showing pGL3-E-cadherin-1200 point-mutations (MUT-1116[T > G], MUT-1115 [T > G], MUT-1114 [A > G], MUT-1113 [A > G], MUT-1112 [A > G], and MUT-1111 [A > G]). (I) Luciferase assay results showing that each nucleotide responded to the transcriptional activation. (J) ChIP results showing interaction of ETV6-MECOM with different E-cadherin promoters. Data were presented as mean ± SEM. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 and ns: not significant.

Fig 4: The cell proliferation curves of the A172 and YKG-1 cells transduced with siRNA against EVI1 (siEVI1_B and siEVI1_C), compared with those of the negative control (siRNA-control). On day 0, the cells were transfected with siRNA. Cell counting was carried out at 24, 48, and 72 h after siRNA transfection. Data shown are the mean ± SD of the quadruplicate analyses. Statistical significance was determined by the Student’s t test, *P < 0.05

Fig 5: EVI1 is phosphorylated at S436.a Schematic illustration of the EVI1 and ΔEVI1-protein isoforms showing DNA binding zinc finger domains (ZF) and CtBP-binding motifs (CtBP). S436 phosphorylation shown in a red circle, in relation to the EVI1-SPLQ motif EVI1 with other species and the putative nuclear localization signal (NLS). Immunoprecipitation of endogenously expressed EVI1 is from SB1690CB cells shown in the western blot of EVI1. b, c Mass spectrometry analysis of the EVI1 peptide Asp433-Lys447 from SB1690CB AML cells non-phosphorylated (b) and S436-phosphorylated peptides (c).