Fig 2: E-boxes function as cis-elements for the EIF4EBP3 promoter activation by TFE3.(a) Sequence (−163 to +59) in the EIF4EBP3 promoter region. Position +1 indicates the information of putative transcription start site (the DBTSS (http://dbtss.hgc.jp)). Protein coding region is shown by red characters. (b) E-box mutation reduces EIF4EBP3 promoter activity. E-box sequences 5′-CAGCTG CACGTG-3′ were changed to 5′-GTCCTG GTGGTG-3′. MiaPaCa-2 cells were transiently transfected with p4E-BP3pro-Luc (−163 to +59) plasmid or its E-boxes mutant and pRL-TK reporter plasmid as a control. After 24 h, cells were treated with 100 nM of INK1341 for 24 h. Relative luciferase activity was measured using Dual-Luciferase Assay kit. Error bars indicate±s.d. (n=3). **P<0.01. Statistical significance was determined using two-way analysis of variance (ANOVA). A representative result of three independent experiments is shown. (c) TFE3 and TFEB expression in three cell lines. Each mRNA expression was analysed by RT–PCR. (d) MiaPaCa-2 cells were transiently transfected with the indicated amount of TFE3 (HA-tag) plasmid. Detection of the proteins was performed by immunoblotting. A representative result of two independent experiments is shown. (e) MiaPaCa-2 cells were transiently transfected with the indicated amount of TFE3 (HA-tag) plasmid together with reporter plasmids as shown in b. After 48 h, Relative luciferase activity was measured using Dual-Luciferase Assay kit. Error bars indicate±s.d. (n=3). **P<0.01. Statistical significance was determined using two-way ANOVA. A representative result of three independent experiments is shown.