Figure 5 from E3 Ubiquitin Ligase Mind Bomb 1 is a Novel Negative Regulator of Tumor-Suppressive BMP Signaling

<jats:p>&lt;p&gt;Loss of MIB1 amplifies BMP target gene expression. &lt;b&gt;A,&lt;/b&gt; Overview of the BMP inhibitor washout experiment for RNA-seq. MIB1 KO was induced by Dox (0.5 μg/mL) under BMP-off conditions (+LDN, 50 nmol/L). After 3 days, cells were split equally into medium containing BMP4 (10 ng/mL) with or without continued LDN (50 nmol/L) treatment, generating groups 1 to 4 as indicated. RNA was harvested 24 hours later for sequencing analysis. &lt;b&gt;B,&lt;/b&gt; In parallel with RNA collection, BMP pathway activity was assessed by IB detection of SMAD1/5/9 phosphorylation 24 hours after washout of the BMP inhibitor. This confirmed effective pathway suppression and reactivation. &lt;b&gt;C,&lt;/b&gt; RNA-seq analysis of gene expression changes following BMP inhibitor washout (&lt;i&gt;n&lt;/i&gt; = 3 per group; 16,400 genes analyzed). The dashed line indicates genes with equivalent FC upon BMP reactivation (BMP-on/BMP-off) in both MIB1 KO and MIB1 WT cells. Potentiated BMP target genes are labeled in bold orange; other BMP target genes and pathway components are indicated with blue labels. &lt;b&gt;D,&lt;/b&gt; Zoomed-in view of the region in &lt;b&gt;C&lt;/b&gt; highlighting potentiated genes (orange), with BMP target genes in bold orange. &lt;b&gt;E,&lt;/b&gt; GSEA of RNA-seq data from the BMP inhibitor washout experiment (&lt;b&gt;C&lt;/b&gt; and &lt;b&gt;D&lt;/b&gt;), comparing group 4 (MIB1 KO, BMP-on) with group 2 (MIB1 WT, BMP-on). Gene sets were derived from the Hallmark database. [&lt;b&gt;A,&lt;/b&gt; Created in BioRender. Cottonham, C. (2026) &lt;a target="_blank" href="https://biorender.com/5qnb2jv"&gt;https://BioRender.com/5qnb2jv&lt;/a&gt;.]&lt;/p&gt;</jats:p>