Fig. 4: MYC activates EIF4EBP1 promoter activity and transcription in MBs.

A ChIP peak locations within the human EIF4EBP1 promoter, exon 1 and part of intron 1 (hg38; Chr8: 38,030,342 - 38,031,906) from ChIP-sequencing data for MYC (Encode consortium, Encyclopedia of DNA Elements at UCSC [57, 58]) and an illustration of the luciferase reporter construct containing the EIF4EBP1 promoter, exon 1 and part of intron 1 (−192; +1372) coupled to Firefly luciferase, with the indicated binding sites of the transcription factor MYC. The three E boxes present in the promoter, and corresponding introduced mutations, are indicated. B, C HEK293-T cells were transfected with the (−192; +1372) EIF4EBP1 promoter reporter construct, together with 25 ng, 50 ng and 100 ng MYC (B) or the (−192; +1372) EIF4EBP1 promoter reporter constructs containing a mutation of each of the E boxes (as indicated in A), together with 25 ng MYC (C). For (B) and (C), a Renilla Luciferase vector was used as an internal control and luciferase activities were detected using the Dual-Luciferase Reporter Assay. Firefly luciferase activity was normalized to Renilla luciferase activity and the ratio was normalized to the corresponding 0 ng (B) or control (C) condition. Data represent the mean of four (B) or three (C) independent replicates ± standard deviation (SD). Significance was calculated using an unpaired and two-tailed parametric t test (*p < 0.05, ****p < 0.0001). A representative immunoblot analyzing expression of MYC is presented in (B). D, E Med8A (D) and HD-MB03 (E) MB cells were transiently transfected with negative control siRNAs (siCtrl), or two different siRNAs each targeting MYC (siMYC#1 and siMYC#2). Cells were re-transfected after 96 h with their corresponding siRNA and incubated for a total of 168 h. MRNA was harvested to determine the expression levels of EIF4EBP1 and MYC by qRT-PCR. Data obtained by qRT-PCR represent the mean of three independent replicates ± SD and the fold change in expression was normalized to the negative control (siCtrl). Significance was calculated using an unpaired and two-tailed parametric t test (*p < 0.05, **p < 0.01, ****p < 0.0001). F Med8A MB cells were transiently transfected with negative control (siCtrl) or a pool of four different siRNAs (see Table S5) targeting MYC (siMYC). Cells were incubated 72 h and protein was harvested for immunoblotting using the indicated antibodies. G, H Control and MYC overexpressing (MYC OE) ONS76 (G) or UW228.3 (H) cells were lysed. Levels of EIF4EBP1 mRNA were determined by qRT-PCR. Levels of 4EBP1 and MYC proteins were determined by immunoblots using the indicated antibodies. Data obtained by qRT-PCR represent the mean of three independent replicates ± SD and the fold change in expression was normalized to the control. Significance was calculated using an unpaired and two-tailed parametric t test (***p < 0.001, ****p < 0.0001).