Fig 2: SMURF2 regulates the ubiquitination of ID2. (A) The expression level of ID2 was detected following SMURF2-KD in A549 cells. (B, C) Detection ID2 protein level in the presence of CHX following SMURF2-KD in H460 cells, and the quantitative result was presented. (D, E) The ubiquitination level of ID2 was detected in HEK293T cells and further detected with the use of MG132. (F) WB analysis of the ubiquitination types of ID2. ***P < 0.001.

Fig 3: SMURF2 depletion promotes the formation and development of xenograft tumors. (A) Tumor volumes were measured and calculated every two days between SMURF2-NC and SMURF2-KD groups. (B) Images of tumor-bearing mice were captured from SMURF2-NC and SMURF2-KD groups. (C) Representative images of tumors obtained from SMURF2-NC and SMURF2-KD groups. (D) Tumor weight was assessed at the end time points the experiments. (E) Typical immunohistochemical images of tumor tissues exhibited the expression of SMURF2, ID2 and p21. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 4: ID2 inhibits E2A-mediated transcriptional activation of p21 expression. (A) Venn diagram was drawn to identify the possible interacted genes of ID2. (B) The Common genes derived from Venn were used to perform KEGG enrichment analysis. (C, D) Endogenous interaction between ID2 and E2A was detected in A549 and H460 cells. (E) The interaction domain between ID2 and E2A was identified by WB analysis. (F, G) p21 expression was detected following E2A OE in A549 and H460 cells. (H) Relative luciferase activity of CDKN1A promoter region in A549 (left) and H460 (right) cells. (I) Evolutionarily conserved E-box binding sites. (J) The predicted E2A binding site in the CDKN1A locus. (K) ChIP assay was used to illustrate the transcriptional regulation of CDKN1A. **P < 0.01; ***P < 0.001.