Fig 2: ID2-mediated MMP2 expression is required for the GDF-11-stimulated human EVT cell invasion. A and B, HTR-8/SVneo (A) and primary EVT (B) cells were treated with 30 ng/mL GDF-11 and the cell invasiveness was measured by the Matrigel transwell invasion assay. C, HTR-8/SVneo cells were transfected with 50 nM control siRNA (si-Ctrl) or MMP2 siRNA (si-MMP2) for 48 h, and then treated with 30 ng/mL GDF-11 (G11) for 24 h. The protein levels of MMP2 were examined by western blot. D and E, HTR-8/SVneo cells were transfected with 50 nM control siRNA (si-Ctrl), MMP2 siRNA (si-MMP2) (D), or ID2 siRNA (si-ID2) (E) for 48 h, and then treated with 30 ng/mL GDF-11 (G11). The cell invasiveness was measured by the Matrigel transwell invasion assay. For the invasion assay, the top panel shows representative photos of the invaded cells. The bottom panels show summarized quantitative results. The results are expressed as the mean ± SEM of at least three independent experiments. Multiple comparisons were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. Values that are statistically different from one another (p < 0.05) are indicated by different letters. The values with any common letter are not significantly different

Fig 3: Induction of ID2 is required for the GDF-11-induced MMP2 expression in human EVT cells. A and B, HTR-8/SVneo and primary EVT cells were treated with 30 ng/mL GDF-11 (G11) for 1, 3, and 6 h. The mRNA (A) and protein (B) levels of ID2 were examined by RT-qPCR and western blot, respectively. C, HTR-8/SVneo cells were transfected with 50 nM control siRNA (si-Ctrl), SMAD2 siRNA (si-SMAD2), or SMAD3 siRNA (si-SMAD3) for 48 h, and then treated with 30 ng/mL GDF-11 (G11) for 3 h. The protein levels of ID2, SMAD2, and SMAD3 were examined by western blot. D, Cells were transfected with 50 nM control siRNA (si-Ctrl) or ID2 siRNA (si-ID2) for 48 h, and then treated with 30 ng/mL GDF-11 (G11) for 24 h. The protein levels of MMP2 and ID2 were examined by western blot. The results are expressed as the mean ± SEM of at least three independent experiments. Multiple comparisons were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. Values that are statistically different from one another (p < 0.05) are indicated by different letters. The values with any common letter are not significantly different

Fig 4: Loss of Id2 increased apoptosis in the intestinal crypt of Apc?716 mice. (A-D) Apoptotic cells (closed arrowheads) in (A) the ileal crypts and (C) villi of representative Apc?716 and Apc Id2 mice, visualized by immunohistochemical analysis using an antibody against cleaved caspase-3. Closed arrowheads, cleaved caspase-3 positive epithelial cells; open arrowhead, cleaved caspase-3 positive stromal cell. Scale bars, 25 µm and 50 µm (A and C, respectively). Quantification of apoptotic cells in the (B) ileal crypts and (D) villi of Apc?716 and Apc Id2 mice. (E) Cell proliferation in the ileal crypts of representative Apc?716 and Apc Id2 mice, visualized by immunohistochemical analysis using an anti-Ki67 antibody. Scale bars, 25 µm. (F) Quantification of Ki67-positive cells in the ileal crypts of Apc?716 and Apc Id2 mice. The results are given as means±s.d. (n=3). *P<0.05.

Fig 5: Increased cytosolic localization of Id2 characterizes ILC.A–C Cytosolic Id2 expression is prevalent in ILC. Shown are representative tissue micro array (TMA) cores (top panels) from IDC-NST and ILC stained for Id2 expression (A) (brown). A total of 148 invasive breast cancer samples were scored as binary output and quantified in B. Note the overall increase in Id2 expression when comparing ILC versus IDC-NST in B, and the increase in the number of cases that express cytosolic Id2 when comparing ILC versus IDC-NST (C). Pearson Chi-square tests were used to determine statistical significance. P-values are stated in the table. D Shown are Id2 mRNA expression levels in the TCGA dataset. Wilcoxon rank tests were used to assess statistical significance. In total 533 breast cancer samples were analyzed (388 IDC-NST versus 145 ILC). E Id2 controls sustained indolent proliferation in ILC. Cartoon depicting a proposed model for E-cadherin dependent regulation of cell cycle progression. Show are E-cadherin expressing (left panel) and E-cadherin negative breast cancer (right panel). Note the consequences of E-cadherin loss (right panel), inducing cytosolic and nuclear translocation of p120 and a subsequent de-repression of Id2 expression by Kaiso. Id2 will accumulate in the cytosol, where it binds hypo-phosphorylated Rb and inhibits phosphorylation of Rb by CDK4/6. Cell cycle progression will be dampened, underpinning the indolent and low-grade features of E-cadherin mutant ILC. Under anchorage independent conditions (dissemination), Id2 expression is further increased due to a nuclear influx of p120 [26], leading to a full prevention of Rb phosphorylation, clustering E2F, and inhibition of cell cycle progression in G0/G1.