Fig 2: SMURF2 knockdown promotes cell proliferation and tumorigenesis. (A, B) Cell proliferation was monitored by CCK-8 assay of A549 and H460 cells at the indicated time points (0 h, 24 h, 48 h and 72 h). (C) A549 cell cycle was determined by flow cytometry with quantitative analysis (right panel). (D) H460 cell cycle was determined by flow cytometry with quantitative analysis (right panel). (E) Cell migration of A549 cells were assessed by wound healing assay with quantitative analysis (bottom panel). (F) Cell migration of H460 cells were assessed by wound healing assay with quantitative analysis (bottom panel). (G) Migration and invasion of A549 cells were monitored by transwell assay with quantitative analysis (right panel). (H) Migration and invasion of H460 cells were monitored by transwell assay with quantitative analysis (right panel). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance; scale bar, 100 μm.

Fig 3: SMURF2 expression and its diagnostic and prognostic value in patients with lung cancer. (A) Data from TCGA-LUAD revealed that SMURF2 was downregulated in tumor tissues. (B-D) The expression of SMURF2 in lung tumor tissues compared to adjacent normal tissues in GSE75037, GSE149507, GSE130779 (dropped one case of outliers). (E) SMURF2 expression based on tumor stage of patients with LUAD. (F) Kaplan-Meier curve was performed to analyze the prognosis value of SMURF2 in patients with LUAD. (G) SMURF2 expression as well as several key clinical covariates were analyzed by cox proportional hazard model based on TCGA lung adenocarcinoma (n = 508 patients). *P < 0.05; **P < 0.01.

Fig 4: 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 5: The effects of SMURF2 on the expression of cell cycle proteins in lung cancer cell lines. (A) Biological processes enrichment analysis of the target genes co-expressed with SMURF2. (B-D) RT-qPCR and WB analysis of SMURF2 expression in SMURF2-KD A549 (b) and H460 (c) cells. (E, F) Detection of mRNA level of p18, p21, p27, CDK2, CDK4 and CDK6 in SMURF2-KD A549 (e) and H460 (f) cells. (G, H) SMURF2-OE efficiency of A549 cell was detected by RT-qPCR (g) and WB (h). (I) Detection of mRNA level of p21, p27, CDK2 and CDK4 in SMURF2-OE A549 cells. (J, K) SMURF2-OE efficiency of H460 cell was detected by RT-qPCR (j) and WB (k). (L) Detection of mRNA level of p21, p27, CDK2 and CDK4 in SMURF2-OE H460 cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance.