Figure 2 from E3 Ubiquitin Ligase Mind Bomb 1 is a Novel Negative Regulator of Tumor-Suppressive BMP Signaling

<jats:p>&lt;p&gt;BMP ligands, but not TGF-β, inhibit growth of cell lines dependent on both MIB1 and FKBP1A. &lt;b&gt;A,&lt;/b&gt; Gene dependency scores for MIB1 and FKBP1A plotted for 1,164 cancer cell lines in DepMap (Public 22Q4 Chronos). Cell lines dependent on MIB1 or FKBP1A were defined by a dependency score ≤ −0.5. &lt;b&gt;B,&lt;/b&gt; MIB1- and FKBP1A-dependent cell lines were pretreated with BMP receptor inhibitor LDN (50 nmol/L) for 15 minutes followed by treatment with a cocktail of BMP ligands (top) or pretreated for 15 minutes with TGF-β receptor inhibitor SB431542 (SB, 5 μmol/L) followed by treatment with a TGF-β ligand cocktail (bottom). After 6 days, a CellTiter-Glo luminescent assay was performed. Statistical significance shown for the comparison between cytokine-treated conditions (+BMP4 or +TGF-β1) and the control group (two-way ANOVA with Tukey test, 4-group comparison). &lt;b&gt;C,&lt;/b&gt; IncuCyte live-cell analysis over 6 days for NCIH838 cells treated with the BMP (left) or TGF-β (right) cocktails in the presence or absence of 50 nmol/L LDN or 5 μmol/L SB, respectively. &lt;b&gt;D,&lt;/b&gt; CellTiter-Glo assay of NCIH838 cells treated with a titration of the indicated ligands over 6 days. All conditions were included in the two-way ANOVA with Dunnett’s test; statistical significance is indicated only for comparisons of +BMP4 vs. −BMP4 without LDN. &lt;b&gt;E,&lt;/b&gt; IncuCyte live-cell analysis for NCIH838 cells treated with 50 nmol/L LDN, 10 ng/mL of BMP4, or both. &lt;b&gt;F,&lt;/b&gt; NCIH838 cells were treated with 50 nmol/L LDN, 10 ng/mL of BMP4 or both for 7 days and then assayed by CellTiter-Glo. The results are represented as the mean ± SD; &lt;i&gt;n&lt;/i&gt; = 3 (two-way ANOVA with Tukey correction). &lt;b&gt;G&lt;/b&gt; and &lt;b&gt;H,&lt;/b&gt; Colony formation assays for NCIH838 cells treated with titrations of BMP4 in the absence (control) or presence of LDN (&lt;b&gt;G&lt;/b&gt;) or TGF-β1 in the absence (control) or presence of SB (&lt;b&gt;H&lt;/b&gt;) for 7 days; scale bars, 5 mm. Representative wells shown; the experiment was repeated independently at least three times. &lt;b&gt;I,&lt;/b&gt; IF staining of NCI-H838 cells for phosphorylated SMAD proteins (orange). pSMAD1/5/9 staining under control (untreated) conditions or after treatment with BMP4 (11 ng/μL), with or without 50 nmol/L LDN. pSMAD2/3 staining under control conditions or after treatment with TGF-β1 (11 ng/μL), with or without 5 μmol/L SB. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. &lt;b&gt;J,&lt;/b&gt; Immunoblot of cytoplasmic (C) and nuclear (N) fractions from NCI-H838 cells pretreated with 1 μmol/L LDN or 5 μmol/L SB for 24 hours and then stimulated with 10 ng/mL BMP4 or TGF-β1 for 1.5 hours to detect pSMAD proteins. CREB and HSP90 were used as loading controls for nuclear and cytoplasmic fractions, respectively. For all panels, *, &lt;i&gt;P&lt;/i&gt; &lt; 0.05; **, &lt;i&gt;P&lt;/i&gt; &lt; 0.01 and ***, &lt;i&gt;P&lt;/i&gt; &lt; 0.001; ****, &lt;i&gt;P&lt;/i&gt; &lt; 0.0001; “ns,” not significant.&lt;/p&gt;</jats:p>