Fig 2: RICTOR degraded TELO2 by ubiquitination under serum deprivation. (A) LoVo cells were transfected with scrambled siRNA or RICTOR siRNA and cultured with or without serum for 24 h. CHX (50 µg/ml) was added to block protein synthesis at different time points, and the protein levels of TELO2 or RICTOR were examined by western blot analysis. (B) ImageJ software was used to scan the grey scores of TELO2 expression, and a line chart was drawn to present the change in the difference of TELO2 expression between the groups. (C) LoVo cells containing scrambled siRNA or RICTOR siRNA were transfected with His-ubiquitin. Forty-four hours post-transfection, cells were incubated in serum-deprived conditions for an additional 24 h, and then 1% triton lysis buffer was used to lysate the cells. Immunoprecipitation analysis was performed using a TELO2 antibody, and normal IgG was used as a negative control. (D) LoVo cells were incubated with or without serum for 24 h, and then 1% triton lysis buffer was used to lysate the cells. Immunoprecipitation analysis was performed using a RICTOR antibody, and normal IgG was used as a negative control. All of the experiments were performed twice. (E) The graphical abstract clarified the role of TELO2 by RICTOR in CRC progression under different conditions.

Fig 3: Knockdown of RICTOR inhibited TELO2-induced tumorigenesis in CRC. Stable Myc-TELO2 LoVo cells were transfected with RICTOR-siRNA. The experimental groups, including ctrl, Myc-TELO2, and Myc-TELO2 plus RICTOR-siRNA, were defined as groups 1, 2, and 3, respectively. (A) A WST-1 assay was used to detect the proliferation rate at different times (*P<0.05). (B) A soft agar assay was preformed to detect tumorigenic ability. The number of colonies for the different transfections are shown below the panel (*P<0.05). (C) Flow cytometry was performed to investigate the cell cycle distribution, and the right panel shows the percentage of cells in the different stages. (D) Wound healing assay was used to investigate the migration ability. The numbers mark the interval distance after scratching at the indicated time points. (E) A Transwell chamber assay was used to detect the invasion ability, and the numbers below indicate the invaded cells in each chamber. All of the experiments were performed three times.

Fig 4: TELO2 was expressed at high levels in colorectal cancer (CRC) cells. (A) The expression pattern of TELO2 mRNA in normal (N) and cancerous (C) tissues of the digestive system. The boxes represent median expression, and the dots represent outliers. *Significant increase in TELO2 expression as compared with the indicated paired tissue. #No significant difference between the pair. (B) Four pairs of resected tumors and adjacent non-tumor tissue specimens were subjected to protein extraction and western blot analysis. N represents normal tissues, and C represents cancer tissues. (C) In two selected CRC patients, there was a higher expression of TELO2 in cancer tissues as confirmed by IHC. The magnification of the image is ×200, and the scale bars are 100 µm. (D) The expression difference between cancer tissues (n=100) and non-cancerous tissues (n=80) (*P<0.001). (E) Log-rank analysis was used to detect the survival difference between TELO2-positive and -negative patients (P=0.216).

Fig 5: RICTOR bound to TELO2, and its expression was positively correlated with TELO2 in CRC. (A) Potential TELO2-binding partners were predicted using a STRING database. Red boxes represent protein-protein interactions. (B) LoVo cells were transfected with Myc-TELO2 or pLPC-Myc. CHAPS lysis buffer was used to prepare whole cell lysates, and immunoprecipitation was performed to detect the interaction between TELO2 and RICTOR. The experiments were repeated twice. (C) An immunohistochemistry assay was performed to investigate TELO2 and RICTOR expression in serial sections of cancer tissue from one patient (×200). The red arrow shows high expression of both TELO2 and RICTOR, and the blue arrow shows low expression of both TELO2 and RICTOR. (D and E) Spearman correlation analysis was performed to test the expression relevance between TELO2 and RICTOR in CRC tissues (n=100) and adjacent normal tissues (n=77). (F) A heat map shows the correlation between TELO2 and RICTOR expression.