Fig. 6: PGAM5 promotes CRC progression through its phosphatase activity.

a, b TCGA database analysis showed that PGAM5 expression was upregulated in both COAD (Colon adenocarcinoma, a) and READ (Rectum adenocarcinoma, b). n: sample number of patients per colorectal tumor type. c Representative immunohistochemical images of PGAM5 in human colorectal peri-tumor and tumor tissues from CRC patients. Scores of PGAM5 expression levels were quantified. n = 49. Scale bar: 100 µm. P = 0.0003. d qRT-PCR analysis for PGAM5 in human colorectal peri-tumor (n = 13) and tumor tissues (n = 9) from CRC patients. P = 0.006. e Western blotting for PGAM5 in human colorectal peri-tumor and tumor tissues from CRC patients. β-Actin was used as the loading control. Same blot for β-Actin as shown in Supplementary Fig. 4 g. f Gross images of AOM-DSS tumors and quantification of tumor numbers (P = 0.0008) per mouse and tumor area (P = 0.013) in Ctrl and Pgam5^cKO mice. Ctrl: n = 39 tumors from 4 mice; Pgam5^cKO: n = 15 tumors from 4 mice. g Double immunofluorescence of Ki67 and β-Catenin in colon tumors from Ctrl and Pgam5^cKO mice. n = 50 crypts from 4 mice. Percentage of Ki67⁺ cells in tumors was quantified. Scale bar: 100 µm. h Gross images of xenografted tumors 4 weeks after transplantation with pcDNA3.1, pcDNA3.1-PGAM5^WT or pcDNA3.1-PGAM5^H105A–transfected HCT116 cells. Tumor weight was quantified. n = 5 mice. P = 0.0375; P = 0.0001. i Immunofluorescence of EdU in LOVO cells after transfection with pcDNA3.1, pcDNA3.1-PGAM5^WT or pcDNA3.1-PGAM5^H105A plasmids. The percentage of EdU⁺ cells was quantified. n = 29 fields from 3 independent samples. Scale bar: 25 µm. P = 0.0001. j Colony formation assay was used to detect the effect of PGAM5 phosphatase activity in LOVO cells after transfection with pcDNA3.1, pcDNA3.1-PGAM5^WT or pcDNA3.1-PGAM5^H105A plasmids. n = 3 independent samples. k, l Transwell assay was used to detect migration (k, P = 0.0087) and invasion (l, P = 0.0042) of LOVO cells transfected with pcDNA3.1, pcDNA3.1-PGAM5^WT or pcDNA3.1-PGAM5^H105A plasmids. n = 3 independent samples. Scale bar: 250 µm. Western blotting images are representative of experiments that were repeated at least three times with similar results. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Statistical analysis in panels h, i, k, and l was performed with one-way ANOVA followed by Tukey’s test, and the rest were performed with two-tailed unpaired Student’s t-test.