Fig 1: MIB1 negatively regulates BMPR2 protein levels. A, Global proteomic analysis by mass spectrometry of MIB1-iKO cells treated with Dox (0.5 μg/mL) for 3 days (n = 3). Volcano plot displays proteins with >2-fold change and statistical significance based on −log10 (P value) for MIB1 KO (+Dox) vs. MIB1 WT (−Dox). A total of 7,860 proteins were quantified. Orange points indicate proteins with >2-fold change and meeting the significance threshold; blue points indicate statistically significant proteins with <2-fold change; gray points did not meet the significance criteria. B, RNA-seq expression (RPKM) of selected genes from MIB1-iKO cells treated with Dox (0.5 μg/mL) for 3 days under basal BMP signaling conditions. BMPR2 and ACVR1 mRNA levels do not show notable changes. Each plot shows two biological replicates per condition (n = 2). FC was calculated from RPKM values comparing +Dox, KO vs. −Dox, WT. No statistical analysis was performed. RPKM, normalized reads per kilobase gene model per million total reads. C, Immunoblot analysis of BMP pathway proteins in MIB1-iKO cells treated with Dox (0.5 μg/mL) for 3 days. β-Actin served as the loading control. D, Immunoblot analysis of biotinylated cell surface proteins from MIB1-iKO cells treated with or without Dox (0.5 μg/mL) for 4 days, showing BMPR2 at the plasma membrane in the absence of MIB1. Immunoblot (E) and qPCR (F) analyses for HUVECs transfected with siRNA targeting MIB1, HPRT (positive control), or a nontargeting control (NTC) for 72 hours. RNA expression normalized to GAPDH. Experiments were repeated with at least three donors. P values were calculated using an unpaired Student t test; ****, P < 0.0001; and “ns,” not significant.
Fig 2: MIB1 and BMPR2 associate in cancer and endothelial cells. A, Co-IP of endogenous BMPR2 and MIB1 from NCIH838 cells treated with CLQ (25 μmol/L) or MG132 (10 μmol/L) for 6 hours prior to lysis to stabilize BMPR2 protein levels. IB confirmed co-precipitation of MIB1. Dox, used to induce KO of MIB1. B, Co-IP of endogenous BMPR2 and MIB1 from HUVECs transfected with control or BMPR2 siRNA for 72 hours. IP and IB were performed as described in A. C, Schematic (top) illustrates the experimental workflow for pull-down of endogenous MIB1 from HUVEC lysates using recombinant His-tagged BMPR2. Association of MIB1 with BMPR2 was verified by IB.
Fig 3: The ligase domains of MIB1 are essential for its regulatory function in BMP. A, Top, schematic (modified from (20)) of the endogenous MIB1 coding region, with exons shown as boxes and protein domains indicated. A, Bottom, Cas9-resistant MIB1 cDNA constructs, containing mutated PAM sequences (asterisks), express either WT MIB1 cDNA (MIB1 WT, blue) or MIB1 cDNA with all three RING domains deleted (MIB1ΔR1-3, orange). B, Immunoblot analysis of MIB1 protein levels in MIB1-iKO cells that were engineered to stably express Cas9-resistant MIB1 WT or MIB1 ΔR1-3 after 3 days of Dox (0.5 μg/mL) treatment. β-Actin served as the loading control. C–E, MIB1-iKO cells overexpressing MIB1 WT or MIB1ΔR1–3 were treated with Dox (0.5 μg/mL) for the indicated time points. C, Colony formation assay after 14 days of Dox treatment; scale bars, 10 mm. Images are representative of three independent experiments. D, CellTiter-Glo assay after 7 days of Dox treatment. Data represent three independent experiments. Two-way ANOVA with Tukey test; *, P < 0.05. “ns” denotes nonsignificant differences between no MIB1 cDNA cells and those expressing MIB1 constructs. E, Immunoblot analysis of BMPR2 immunoprecipitates from MIB1-iKO cells. Lane labels indicate MIB1 WT (no cDNA, −Dox), MIB1 KO (no cDNA, +Dox; Dox-induced KO of endogenous MIB1), MIB1 WT OE (MIB1-mNeonGreen expressed in MIB1 KO cells), and MIB1ΔR1-3 OE (MIB1ΔR1-3-IRES-mNeonGreen expressed in MIB1 KO cells). Cell lysates were subjected to IP with anti-BMPR2 antibody or IgG control, followed by IB with the indicated antibodies. Input lysates are shown for comparison. A longer exposure of the MIB1 immunoblot is included to facilitate detection of MIB1 in BMPR2 immunoprecipitates. Blue and orange arrowheads indicate BMPR2-associated MIB1 WT OE and MIB1ΔR1-3 OE, respectively. β-Actin is shown as an input and specificity control. F, Densitometric quantification of MIB1 and BMPR2 protein levels from the input immunoblots shown in E. Signals were normalized to β-Actin and expressed relative to the MIB1 WT (no cDNA, −Dox) control.
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