PADI3 induces cell cycle arrest via the Sirt2/AKT/p21 pathway and acts as a tumor suppressor gene in colon cancer

Effects of PADI3 on HCT116 and LoVo cells. RFP-expressing cells were used as controls. (A) Weak PADI3 expression was detected in HCT116 and LoVo cells. (B) Western blot analysis was used to measure PADI3 and RFP expression in both LoVo and HCT116 cells. (C, D) A CCK-8 assay measured the proliferation of HCT116 and LoVo cells. (E) The colony formation ability of HCT116 cells was measured and statistically analyzed using a colony formation assay following 14 days of culture. (F) The colony formation ability of LoVo cells was measured and statistically analyzed using a colony formation assay following 14 days of culture. * indicates P < 0.05 for three independent experiments analyzed by Student’s t-test.

Effects of PADI3 in vivo. (A) Western blot analysis was used to measure PADI3 expression. (B) PADI3-expressing HCT116 cells were injected into the left dorsal flank of BALB/c nude mice, and RFP-expressing HCT116 cells were injected into the right dorsal flank. (C) Tumors were dissected 42 days after cell injection. (D) Tumor weight was measured and statistically analyzed. (E) The tumor formation ratio was statistically analyzed. * indicates P < 0.05.

Analysis of the roles of PADI3 in the cell cycle using FCM. (A) FLAG-expressing HCT116 cells were used as the negative control group. (B) PADI3-FLAG-expressing HCT116 cells were used as the experimental group. (C) Statistical analysis of A and B. (D) FLAG-expressing LoVo cells were used as the negative control group. (E) PADI3-FLAG-expressing LoVo cells were used as the experimental group. (F) Statistical analysis of D and E.

RNA-sequencing analysis of PADI3-expressing HCT116 cells. (A) Heat map from the RNA-sequencing analysis. (B) Expression of phosphorylated AKT (p-AKT), AKT, Sirt2, Snail and p21 in OE-PADI3 HCT116 cells, OE-RFP HCT116 cells is selected as the control group. (C) Western blot analysis was used to measure the expression level of Sirt2 and PADI3 in colon cancer tissues and corresponding tissues. (D) Statistical analysis of the expression level of Sirt2 and PADI3. * indicates P < 0.05 for three independent experiments analyzed by Student’s t-test.

Functional analysis of different PADI3 domains. (A) Predicting the PADI3-domain structure using SMART. (B) Western blot analysis was used to detect the expression of different PADI3 domains in HCT116 cells. (C) CCK-8 assay was used to measure the cell proliferation activity of different PADI3 domains. * indicates P < 0.05 for three independent experiments analyzed by Student’s t-test.

Subcellular locations of the truncated forms of PADI3 in HCT116 cells. (A) Red fluorescence indicates RFP or PADI3-fused RFP, and blue indicates nuclei stained with DAPI. The merged image shows both RFP fluorescence and DAPI staining. The scale bar represents 20 µm. (B) Western blot analysis was used to detect truncated PADI3 in the cytoplasm and nucleus. GAPDH was used as the internal control for cytoplasmic expression, and histone 3 was used as the internal control for nuclear expression. Nu: nucleus, Cy: cytoplasm.

Effects of Sirt2 on PADI3-expressing HCT116 cells. PADI3-expressing HCT116 cells were transfected with pCDNA3.1-Sirt2-iso1 plasmids (OE-PADI3-Sirt2-iso1) or pCDNA3.1-Sirt2-iso2 plasmids (OE-PADI3-Sirt2-iso2) separately. PADI3-expressing HCT116 cells were used as the positive control, and RFP-expressing HCT116 cells were used as the negative control. (A) Western blot analysis was used to measure the expression of AKT, p-AKT, Snail and p21. (B) A CCK-8 assay was used to measure cell proliferation. (C) The cell cycle was analyzed using FCM. (D) Statistical analysis of C. (E) Colony number and size were analyzed. (F) Statistical analysis of E. * indicates P < 0.05 for three independent experiments analyzed by Student’s t-test.