Fig 1: Knockdown expression of PDIA2 inhibited the growth of the subcutaneous xenograft colon cancer. (A) Cell growth analysis of wild-type (Wt), control knockdown and PDIA2-knockdown HT-29 colon cancer cells by Cell Counting Kit-8 assay. (B) Apoptotic cells were costained with annexin-FITC and propidium iodide, and were analyzed by flow cytometry. (C) The follow-up of the xenograft tumor size by a vernier caliper; data were presented as mean ± SD. (D) The xenograft tumors at 27 days after cell transplantation. *, **, and *** represent p < 0.05, 0.01, and 0.001, respectively. ns, not significant.
Fig 2: Knockdown expression of PDIA2 restored metabolic homeostasis in colon cancer cells. (A) The efficiency of PDIA2 knockdown by shRNA strategy. C0 represents control knockdown cells; C1, C2, and C3 indicate the subclone cells with PDIA2 knockdown. C3 was selected for subsequent studies. (B) Glycolysis stress assay of control and PDIA2-knockdown cells using Seahorse extracellular flux (XFe-24) analyzer. 2-DG: 2-Deoxy-D-glucose. (C) Comparison of basal glycolysis (extracellular acidification rate, ECAR), glycolytic capacity, and reserve between control and PDIA2-knockdown cells. (D) Mitochondrial stress assay of control and PDIA2-knockdown cells using a Seahorse extracellular flux (XFe-24) analyzer. FCCP: carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone. (E) Comparison of basal mitochondrial respiration (oxygen consumption rate, OCR) and respiratory reserve between control and PDIA2-knockdown cells. (F) Comparison of proton leak (oxophosphorylation uncoupling) and ATP production between control and PDIA2-knockdown cells. ** and *** represent P < 0.01 and 0.001, respectively.
Fig 3: PDIA 2 expression in human colon cancer and cancer-adjacent tissues. (A) A representative microarray data of 74 pairs of colon cancer and adjacent tissues (left panel) and PDIA2-positive cell counts by Scanscope XT scanning (right panel). (B) The association of PDIA2 overexpression with colon cancer staging and metastasis by a stratified analysis of the microarray results. (C) Western blot analysis of PDIA2 expression in human colon cancer and adjacent colon tissues. T: human colon cancer, A: adjacent tissues. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 4: Coimmunoprecipitation–mass spectrum (Co-IP/MS) analysis of PDIA2-interacting proteins from the HT-29 cell lysate. PDIA2 coimmunoprecipitated with 420 proteins, and the KEGG enrichment was analyzed by STRING software (version 11.0). (A) The overall pathways that were networked by PDIA2. The metabolic pathways were mostly enriched, in which 53 proteins interact with PDIA2. The inset showed the efficient immunoprecipitation of PDIA2 by a gene-specific antibody. IP, immunoprecipitation. (B) Part of protein–interaction network in metabolic pathways; proteins involved in mitochondrial functions were specified in red. (C) The protein–interaction network in endoplasmic reticulum. (D) Western blot analysis of the interaction between PDIA2 and electron transport complexes; labels on the right indicate the subunit of each complex. (E) Confocal microscopy analysis of mitochondrial translocation of PDIA2 in HT-29 colon cancer cells and CCD-18Co human normal colon cells.
Fig 5: Identification of differentially expressed proteins by proteomics in response to AOM/DSS treatment. (A) Display of colon mucosa proteins on two-dimensional electrophoresis gels after saline or AOM/DSS treatment for 7 weeks; the differentially expressed proteins were specified by arrows. (B) Quantitative analysis of the 7 differentially expressed proteins by ImageMaster platinum 6.0 software (GE Healthcare, USA). S: saline, A/D: AOM/DSS. Proteins with greater than two-fold difference (p < 0.05) were selected; one presentative data of three individual experiments was shown. (C) Protein disulfide-isomerase A2 (PDIA2) overexpression in AOM/DSS-treated colon tissues was verified by Western blot. Densitometric analysis of PDIA2 expression was shown in the right panel. (D) Flow chart of 4-phenylbutyric acid (4-PBA) treatment was shown in the upper panel. The changes of the IRE1a phosphorylation status (p-IRE1a), PDIA2, and IRE1a by Western blot analysis were shown as the mean ± SD of individual band intensity in each group in the middle panel, and the counts and sizes of tumors after the treatment of AOM/DSS or the combination of AOM/DSS with 4-PBA were shown in the bottom panel. The upregulations of p-IRE1a and PDIA2 were inhibited by 4-PBA (upper-right panel). Tumors inside the images were indicated with orange arrows; data were shown as the mean ± SD of two individual experiments. *, **, and *** represent p <0.05, <0.01, and <0.001, respectively.
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