Fig 2: JAM3 participated in EMT in colonic epithelial cells.Notes: Molecular interaction networks involving JAM3 (A) and TJP1 (B) were generated by using GCBI database and GeneCards database. (C) Protein expression levels of Bcl2, caspase-3, P21, P27, cyclin D1, E-cadherin, vimentin, TJP1, p-ß-catenin, and ß-catenin in NCM460 cells transfected with siJAM3 and siNC. (D) Protein expression levels of Bcl2, caspase-3, P21, P27, cyclin D1, E-cadherin, vimentin, TJP1, p-ß-catenin, and ß-catenin in JAM3-transfecting as well as control vector transfecting SW620 and HT29 cells.Abbreviations: EMT, epithelial–mesenchymal transition; siNC, siRNA control; GCBI, Gene-Cloud of Biotechnology Information.

Fig 3: JAM3 was hypermethylated in primary CRC tissues, plasma, and cell lines.Notes: (A) Expression level of JAM3 was significantly downregulated in CRC tissues compared to that in normal controls in the Skrzypczak colorectal dataset and TCGA expression array dataset from Oncomine. (B) A significant difference in JAM3 methylation status was observed between 125 CRC tissues and 29 adjacent normal tissues in the GSE25062 dataset. (C) Schematic structure of the JAM3 promoter CpG islands, CpG sites (short vertical lines), and qMSP region is indicated (from forward primer to reverse primer). The transcription start site is shown by a curved arrow. (D) Methylation percentage of JAM3 in 24 paired CRC tissues and normal tissues was detected by qMSP. (E) Methylation percentage of JAM3 in plasma from 18 CRC cases and 18 healthy subjects was examined by qMSP (P<0.001, two-sided Student’s t-test). (F) Methylation percentage of JAM3 in CRC cell lines (HT29, HCT116, SW480, and SW620) and normal colonic NCM460 cells was detected by qMSP (P<0.001, two-sided Mann–Whitney rank-sum test). (G) ROC curves showed the performance of JAM3 methylation status from CRC tissues in predicting CRC. (H) ROC curves showed the performance of JAM3 methylation status from CRC plasma in predicting CRC. Sensitivity means true-positive rate. Specificity means false-positive rate. Results are shown as mean ± SD. ***P<0.001.Abbreviations: CRC, colorectal cancer; TCGA, The Cancer Genome Altas; qMSP, quantitative methylation-specific PCR; ROC, receiver-operating characteristic.

Fig 4: Restoration of JAM3 suppressed CRC cell growth and migration.Notes: (A) Expression of JAM3 in SW620 and HT29 cells transfected with JAM3 expression vector, and control vector was confirmed by Western blot. (B) JAM3 expression significantly suppressed the number of viable cells of JAM3-transfected SW620 and HT29 cells compared to that of control vector-transfected cells (P<0.01, two-sided Student’s t-test). (C) Representative result of transwell assay in JAM3-transfecting as well as control vector transfecting HT29 and SW620 cells. (D) Representative result of monolayer scratch-healing assay in JAM3-transfecting as well as control vector transfecting SW620 and HT29 cells. (E) Representative result of cell-cycle analysis in JAM3-transfecting as well as control vector transfecting SW620 and HT29 cells. Results are shown as mean ± SD. **P<0.01.

Fig 5: JAM3 suppression promoted cell migration and reduced apoptosis in colonic NCM460 cells.Notes: JAM3 mRNA (A) and protein (B) expressions were efficiently inhibited in NCM460 cells transfected with siJAM3. GAPDH was used as an internal control (P<0.01, two-sided Student’s t-test). (C) Representative result of CCK8 assay (P<0.01, two-sided Student’s t-test). (D) Representative colony formation assays in NCM460 cells transfected with siJAM3 and siNC. (E) Representative transwell assays in NCM460 cells transfected with siJAM3 and siNC. (F) Cell-cycle distribution of NCM460 cells transfected with siJAM3 and siNC was detected by flow cytometry analysis. (G) Representative result of cell apoptosis analysis. **P<0.01 and ***P<0.001.Abbreviations: CCK8, Cell Counting Kit-8; siJAM3, siRNA–JAM3; siNC, siRNA control; FITC, fluorescein isothiocyanate.