Fig 2: PTPN18 loss represses the tumorigenicity of HCT116 cells. (A) The sizes of subcutaneous tumors from nude mice were observed and compared. (B) The tumor sizes in the HCT116 PTPN18−/− and control groups were measured using a vernier caliper and recorded regularly. (C) and (D) Tumor tissues from the HCT116 PTPN18−/− and control groups were collected and weighed. (E) PTPN18, MYC and CDK4 protein levels in tumor tissues were assessed by Western blotting. (F) The alterations in CDK4 and MYC protein levels in tumor tissues from the HCT116 PTPN18−/− and control groups were quantified. (G) Histological analysis of tumor tissues was performed using HE staining (Scale bars: 100 μm). Ki-67 staining was examined in tumor tissues using IHC (Scale bars: 100 μm). (H) H-score in IHC assay was calculated. Ki-67 staining was scored as high (3+), moderate (2+), low (1+) and negative (0). The percentage of cells within each tissue core stained at each intensity were recorded to calculate an H-score for each sample. The H-score for staining each sample was defined as: H-score = 0∗ (% at 0) + 1∗ (% at 1+) + 2∗ (% at 2+) + 3∗ (% at 3+). (∗∗, P < 0.01; ∗∗∗, P < 0.001).

Fig 3: MYC interacts with PTPN18, and PTPN18 affects MYC protein levels. (A) Real-time PCR was performed to assess the changes in MYC mRNA expression induced by Flag-PTPN18 overexpression in HCT116 cells. (B) “Substrate-trapping” mutants were constructed, and Co-IP was performed to examine the interaction between Flag-MYC and HA-PTPN18, including mutants in 293T cells. (C) Colocalization was detected between HA-PTPN18D90A/C122S and Flag-MYC in the cytoplasm of HeLa cells through immunofluorescence assays. The Pearson's coefficient of colocalization (r) was 0.56 (Scale bar: 10 μm). (D) The changes in CDK4 and MYC protein levels induced by Flag-PTPN18 overexpression were examined in 293T cells by Western blotting. (E) The alterations in CDK4 and MYC protein levels in 293T cells with Flag-PTPN18 overexpression were quantified. (F) The changes in CDK4 and MYC protein levels were assessed in HCT116 PTPN18−/− and control cell lines by Western blotting. (G) The variation in CDK4 and MYC protein levels in HCT116 PTPN18−/− and control cell lines was quantified. (H) The half-life of MYC in HCT116 PTPN18−/− and control cell lines was assessed by CHX assay. (I) The relative protein levels of MYC in the CHX assay were qualified. (∗∗∗, P < 0.001).

Fig 4: High PTPN18 expression is associated with severe CRC and a low survival rate. (A) The data from the GEO database (GSE4107) was searched to compare PTPN18 mRNA expression in early onset CRC and healthy tissues. (B) mRNA levels of PTPN18 were measured in CRC and paired normal tissues from 8 patients by real-time PCR. (C) A Kaplan–Meier analysis was used to assess the effect of PTPN18 on CRC patient survival. The numbers of CRC patients with high and low PTPN18 expression were both 135. The data were collected from GEPIA (http://gepia.cancer-pku.cn/detail.php?gene=PTPN18). (∗, P < 0.05).

Fig 5: PTPN18 upregulation enhances the proliferation and malignancy of CRC in vitro. (A) The expression of Flag-PTPN18 and Flag-PTPN18D90A C122S was assessed by Western blotting. Lentiviruses were used to construct HCT116 cell lines stably transfected with Flag-PTPN18 and Flag-PTPN18D90A C122S. (B) CCK8 assay was performed to examine the growth and proliferation ability of the Flag-PTPN18-overexpressing, Flag-PTPN18D90A C122S-overexpressing and control cell lines. (C) The proliferation ability of the Flag-PTPN18-overexpressing, Flag-PTPN18D90A C122S-overexpressing and control cell lines was tested through colony formation assays. (D) The cell numbers for the Flag-PTPN18-overexpressing, Flag-PTPN18D90A C122S-overexpressing and control cell lines in the colony formation assay were counted using ImageJ. (E) Soft agar assays were conducted to examine the anchorage-independent growth ability of the Flag-PTPN18-overexpressing, Flag-PTPN18-overexpressing and control cell lines (Scale bar: 200 μm). (F) The statistical analysis of the Flag-PTPN18-overexpressing, Flag-PTPN18D90A C122S-overexpressing and control cell lines for the soft agar assay. (∗, P < 0.05; ∗∗∗, P < 0.001).