Fig 2: Knocking out PTPN18 inhibits the proliferation of HCT116 cells. (A) HCT116 PTPN18-/- cell lines were constructed, and protein expression was detected by Western blotting. (B) CCK8 assays were performed to assess the growth and proliferation ability of the HCT116 PTPN18-/- and control cell lines. (C) Colony formation assays were conducted to examine the proliferation capacity of the HCT116 PTPN18-/- and control cell lines. (D) Cell numbers of the HCT116 PTPN18-/- and control cell lines in the colony formation assays were counted using ImageJ. (E) Soft agar assays were performed to assess the anchorage-independent growth ability of the HCT116 PTPN18-/- and control cell lines (Scale bar: 200 µm). (F) The statistical analysis of the HCT116 PTPN18-/- and control cell lines for the soft agar assay. (**, P < 0.01; ***, P < 0.001).

Fig 3: 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 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 promotes MYC pathway activation. (A) RNA-seq was used to examine the changes in gene expression in HCT116 PTPN18-/- cells compared to that observed in the control cells. (B) Principal component analysis was performed to test the repeatability in groups of HCT116 PTPN18-/- cells and its control cells and the diversity between the two groups. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted to identify the signaling pathways that changed most significantly in the absence of PTPN18. (D) GSEA was performed to assess the mRNA changes of genes in the MYC pathway between the HCT116 PTPN18-/- and control groups. (E) Luciferase assays were carried out to assess the effect of Flag-PTPN18 and Flag-PTPN18D90A C122S overexpression on the MYC pathway in 293T cells. (F) Luciferase assays were conducted to examine the effect of PTPN18 loss on the MYC pathway in HCT116 cells. (G) Real-time PCR was performed to assess the variation of CDK4 mRNA expression in the Flag-PTPN18-overexpressing in 293T cell line. (H) The effect of PTPN18 knockout on CDK4 mRNA levels in HCT116 cells was detected using real-time PCR. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).