Fig. 5

The SMAD9 binding pattern is associated with MYCN. A Venn diagram showing the 7 overlapping downregulated genes between the ChIP-seq and RNA-seq results in BE(2)-C cells. B Q-RT-PCR analyses of the overlapping gene expression after SMAD9 knockdown in MYCN-amp NB cells. C Table showing the expression and dependency profile of 7 overlapping genes based on the DepMap portal. D, E Gene track showing high binding signals for SMAD9 and H3K27ac in the MYCN promoter region detected using ChIP-seq (D). P1, P2 and P3 indicate the primer locations for ChIP-QPCR (D), and the results are shown (E). Sg-1#, 2#, 3# and 4# indicate the sgRNA primers based on the locations for subsequent CRISPRi experiments (D). (F, G) Q-RT-PCR (F) and immunoblotting (G) analyses of MYCN expression after disrupting the binding of SMAD9 to MYCN with the CRISPRi system in MYCN-amp dCas9 STCs. H, I Cell viability on day 6 (H), representative images of the colony formation (I, left panel) and their quantification (I, right panel) for MYCN-amp dCas9 STCs with disrupted binding of SMAD9 to MYCN. J, K SMAD9 rescue experiments showing recovered SMAD9 and MYCN expression detected using Q-RT-PCR (J), and recovered MYCN expression detected using immunoblotting (K). L Relative luciferase activity of the luciferase reporter gene containing the MYCN promoter or SMAD9 enhancer in BE(2)-C cells with SMAD9 knockdown or MYCN knockdown. ChIP: chromatin immunoprecipitation; ChIP-seq: ChIP sequencing; dCas9: dead Cas9; ish: inducible shRNA; MYCN-amp: MYCN amplification; sgSCR: sgRNA scrambled control; shSCR: shRNA scrambled control; STCs: stably transfected cells. *P < 0.05, **P < 0.01, and *** P < 0.001