Fig 2: ID proteins are critical in the BMP9-dependent maintenance of endothelial cell phenotypes. (A–C) RT-qPCR analysis of the siRNA-mediated silencing of endogenous and BMP9-induced Id1 (A), Id2 (B) and Id3 (C) mRNA expression in MS-1 cells. Expression levels were normalized to those of the housekeeping gene Gapdh. Results are expressed as mean ± SD. Representative results from three independent experiments are shown. (D) Fluorescence microscopy analysis of endothelial and mesenchymal markers in MS-1 cells. Non-targeting knockdown, Id1 knockdown, Id2 knockdown and Id3 knockdown MS-1 cells were incubated in medium containing BMP9 (5 ng ml–1) or medium containing ligand buffer (control) for 3 days. The expression of endothelial cell marker PECAM-1 (green) and mesenchymal cell marker SM22α (red) in nuclei (blue) stained MS-1 cells were assessed by using immunofluorescent staining. Scale bar: 50 μm. (E–G) RT-qPCR analysis of the siRNA-mediated silencing of endogenous and BMP9-induced Id1 (E), Id2 (F), and Id3 (G) mRNA expression in MS-1 cells. The expression levels were normalized to those of the housekeeping gene Gapdh. Results are expressed as mean ± SD. Representative results from three independent experiments are shown. (H) Fluorescence microscopy analysis of mesenchymal marker in 2H11 cells. Non-targeting knockdown, Id1 knockdown, Id2 knockdown and Id3 knockdown 2H11 cells were incubated in medium containing BMP9 (5 ng ml–1) or medium containing ligand buffer (control) for 3 days. The expression of mesenchymal cell marker SM22α (red) in nuclei (blue) stained 2H11 cells was assessed by using immunofluorescent staining. Scale bar: 50 μm. (I) Quantified mean fluorescence intensity of SM22α. At least six representative images from three repeated experiments were quantified. Results are expressed as mean ± SD. NS, not significant; **p < 0.005.

Fig 3: Schematic of a working model by which TGF-β and BMP9 regulate EndMT. TGF-β/SMAD signaling induces EndMT by promoting SNAIL expression. BMP9/SMAD signaling promotes SNAIL and ID1/2/3 expression, and stimulates EndMT only when BMP9-induced expression of Id1/2/3 is silenced.

Fig 4: Collaborative mutations of ASXL1 and RUNX1 enhanced ID1 expression. a, b mRNA expression of Id1 was analyzed by RT-qPCR in the spleen (a, left, n = 3), liver (a, right, n = 3), and BMC (b, n = 2) from killed or moribund mice transplanted with control, ASXL1-R693X, RUNX1-R135T, and combined mutations of ASXL1 and RUNX1. Error bars represent the mean ± SD of two repetitions. c Immunoblot analyses of ID1 in the liver and spleen from BMT-mice. d Relative mRNA expression of ID1 in BM leukemic cells from patients harboring either ASXL1-MT (n = 8) or RUNX1-MT (n = 7) and coexisted RUNX1 and ASXL1 mutations (n = 5). Gene expression was measured using real-time qPCR and shown as a relative to normal bone marrow cells (average of 3). NC indicated normal controls. e Immunoblot analyses with indicated antibodies in transduced U937 cells (left), K562 cells (middle), and 32D cells (right); β-actin representing as an internal control. f Knocked down ID1 in the transduced U937 and K562 cells using two different shRNA and scrambled (shLuc), and silencing efficiency was checked by immunoblot analyses after 5 days. g Silenced cells were incubated for 72 h, and cell proliferation was measured by trypan blue exclusion method. Data represented here are the mean ± SD of triplicate cultures, and experiments repeated twice. P value showing in the figure calculated either compared with the control or as indicated in figures. The values (c) indicating relative signal density corresponding to actin expression

Fig 5: Suppression of TCF4 and TWIST1 by ID1 involves TGFβ1−mediated EMT gene expression. (A) Interaction of TCF4 and TWIST1 in the absence or presence of TGFβ1 (20 ng/mL for 24 h). (B) EMT-associated gene expression upon TGFβ1 (20 ng/mL for 24 h) in GFP or ID1 stably expressing A549 cells. (C) TGFβ1−mediated EMT gene expression in control or TCF4 silenced A549 cells. (D) Promoter occupancy of TCF4 on CDH1 and CDH2 promoter in GFP, FOXA1 or PGC1α stably expressing A549 cells. Values represent mean ± SD (n = 3). * p < 0.05 and ** p < 0.01 by one-way ANOVA with Tukey post−test for multiple comparisons. Not significate ns is normally this meaning.