Fig 2: ERp29 coupling with CUL5 protects CRC cells from the ERS-mediated suppression of malignancies. (A) Immunofluorescence images of ERp29 co-locating with CUL5 in CRC cells. (B) Co-immunoprecipitation revealed that ERp29 interacted with CUL5 in CRC cells. (C) Expression of CUL5 in hiERp29 and shERp29 cell lines. (D) Expression of CUL5 and ERp29 in series of tumor sample sections from patients with CRC; scale bar, 50 µm. (E) Expression of CUL5 was upregulated with TM stimulation. (F) hiERp29-siCUL5 and shERp29-siCUL5 cells in ERS. (G) When the hiERp29 cells or shERp29 cells were affected by the knockdown of CUL5 and treated with TM, the migration rates were substantially reduced. Scale bar, 50 µm. (H) Quantified migration rates. Error bars represent the mean ± standard deviation of experiments in triplicate. **P<0.01 with comparisons shown by lines. CRC, colorectal cancer; ERp29, endoplasmic reticulum protein 29; CUL5, cullin 5; TM, tunicamycin; hi-, high; sh-, short hairpin RNA; si-, small interfering RNA; ERS, endoplasmic reticulum stress; 4-PBA, 4-phenylbutyric acid; IgG, immunoglobulin G.

Fig 3: High expression of ERp29 was associated with the poor prognosis of patients with CRC. (A) Immunostaining of ERp29 protein in CRC tissue samples. Scale bar, 50 µm. (B) Kaplan-Meier survival curve for patients with CRC with ERp29. ERp29, endoplasmic reticulum protein 29; CRC, colorectal cancer.

Fig 4: Endoplasmic reticulum stress inhibits the migration and metastasis of CRC cells, but ERp29 offsets these effects. (A) A Transwell invasion assay revealed that the migration rate of cells was reduced in SW620-TM and HCT116-TM cells; correspondingly, the cell migration inhibition receded under supplementation with 4-PBA. TM, 20 µg/ml; 4-PBA, 5 mmol/l. Scale bar, 50 µm. (B) Quantified Transwell invasion assay results. (C) A Transwell invasion assay revealed an increase in cell migration in hiERp29 cells or the decreasing migration of shERp29 cells in comparison with the control when the cells were stimulated with TM. Scale bar, 50 µm. (D) Quantified Transwell invasion assay results. (E) Experimental metastases of nude mice livers and spleen tumors of inoculated CRC cells with histopathological images. Scale bar, 100 and 50 µm. (F) In vivo imaging of mice peritoneal cavities and (G) quantified number of liver metastases. Error bars represent the mean ± standard deviation. *P<0.05 and **P<0.01 with comparisons shown by lines. CRC, colorectal cancer; ERp29, endoplasmic reticulum protein 29; TM, tunicamycin; 4-PBA, 4-phenylbutyric acid; hi-, high; sh-, short hairpin RNA.

Fig 5: ERp29 promotes EMT during ERS depending on the cooperation of CUL5. Western blotting revealed the expression of EMT-associated proteins in (A) colorectal cancer cells under TM stimulation and/or supplemented with 4-PBA, (B) hiERp29 and hiERp29-siCUL5 cells during TM stimulation, and (C) shERp29 and shERp29-siCUL5 cells during TM stimulation. ERp29, endoplasmic reticulum protein 29; EMT, epithelial-mesenchymal transition; CUL5, cullin 5; TM, tunicamycin; hi-, high; sh-, short hairpin RNA; si-, small interfering RNA; 4-PBA, 4-phenylbutyric acid; Snail, snail family transcriptional repressor 1.