Fig 1: Prognostic genes validation with GEPIA and the KM plotter. (A) COL1A1 and PLEK2 had higher expression levels in the LUAD specimen compared to the normal specimen, while GPX3 had the opposite expression in the GEPIA tumor database. T, tumor tissues (red); N, normal tissues (gray). *, P<0.05. (B) The prognostic information of the three genes in LUAD was demonstrated using the KM plotter. The red curve represents the high expression group, and the black curve represents the low expression group. The representative IHC images and prognostic scores of the genes in LUAD vs. adjacent tissue. (C) Expression of PLEK2, COL1A1, and GPX3 in LUAD tissue and adjacent tissue (DAB/hematoxylin staining; magnification 200×). (D) The expression levels of the three genes were analyzed in 98 LUAD tissues and 80 adjacent tissues. PLEK2 and COL1A1 were more highly expressed in LUAD tissue, while GPX3 exhibited an opposite expression trend (P<0.05). LUAD, lung adenocarcinoma; HR, hazard ratio; IHC, immunohistochemistry; GEPIA, Gene Expression Profiling Interactive Analysis; KM, Kaplan-Meier; DAB, diaminobenzidine.
Fig 2: PLEK2 modulates apoptosis in colorectal carcinoma cells. (A) HCT-116 and (B) RKO cells transfected with siCtrl, siPLEK2#1 and siPLEK2#2 were stained with PI/Annexin V-FITC and cell apoptosis was analyzed using a flow cytometry system. Left, apoptosis graphs. Right, quantitative analysis of cell apoptosis. Ctrl and PLEK2-OE (C) HCT-116 and (D) RKO cells were stained with PI/Annexin V-FITC and cell apoptosis was analyzed using a flow cytometry system. Left, apoptosis graphs. Right, quantitative analysis of cell apoptosis. **P<0.01; ***P<0.001 vs. siCtrl or Ctrl. Ctrl, emptry vector; PLEK2-OE, pleckstrin 2 overexpression; si/siRNA, small interfering RNA; siCtrl, negative control siRNA.
Fig 3: PLEK2 modulates cell cycle progression in colorectal carcinoma cells. (A) HCT-116 and (B) RKO cells transfected with siCtrl, siPLEK2#1 and siPLEK2#2 were stained with PI and the cell cycle distribution was analyzed using a flow cytometry system. Left, cell cycle graphs. Right, quantitative analysis of the cell cycle distribution. *P<0.05; **P<0.01 vs. siCtrl. Ctrl and PLEK2-OE (C) HCT-116 and (D) RKO cells were stained with PI and the cell cycle distribution was analyzed using a flow cytometry system. Left, cell cycle graphs. Right, quantitative analysis of the cell cycle distribution. *P<0.05; **P<0.01 vs. Ctrl. Ctrl, empty vector; PLEK2-OE, pleckstrin 2 overexpression; si/siRNA, small interfering RNA; siCtrl, negative control siRNA.
Fig 4: PLEK2 overexpression reinforces cellular functions in colorectal carcinoma cells. (A) Immunoblotting detection of PLEK2 in HIEC, RKO and HCT-116 cells. GAPDH was used as a loading control. Immunoblotting results of PLEK2 in (B) HCT116 and RKO cells transfected with siCtrl, siPLEK2#1 and siPLEK2#2, and (C) in Ctrl- and PLEK2-OE HCT-116 and RKO cells. GAPDH was used as a loading control. Cell proliferation was evaluated in (D) HCT116 and RKO cells transfected with siCtrl, siPLEK2#1 and siPLEK2#2, and (E) in Ctrl- and PLEK2-OE HCT-116 and RKO cells. **P<0.01 and ***P<0.001. Colony formation assay of (F) HCT-116 and RKO cells transfected with siCtrl, siPLEK2#1 and siPLEK2#2, and (G) Ctrl- and PLEK2-OE HCT-116 and RKO cells. **P<0.01 and ***P<0.001 vs. siCtrl or Ctrl. Ctrl, empty vector; PLEK2-OE, pleckstrin 2 overexpression; si/siRNA, small interfering RNA; siCtrl, negative control siRNA.
Fig 5: PLEK2 expression predicts the sensitivity of colorectal carcinoma cells to 5-FU. (A) HCT-116 cells transfected with siCtrl, siPLEK2#1 and siPLEK2#2 were incubated with various concentrations of 5-FU for 48 h. Cell viability was assessed by CCK8 analysis. *P<0.05; **P<0.01 vs. siCtrl. (B) Ctrl and PLEK2-OE RKO cells were incubated with various concentrations of 5-FU for 48 h. Cell proliferation was assessed by CCK8 analysis. *P<0.05; **P<0.01 vs. Ctrl. (C) siCtrl and siPLEK2#2 HCT-116 cells were seeded in 6-well plates at an equal density and incubated with vehicle DMSO or 5-FU for 10 days. Subsequently, colony formation was analyzed. **P<0.01 vs. vehicle. (D) Ctrl and PLEK2-OE RKO cells were seeded in 6-well plates at an equal density and incubated with vehicle or 5-FU for 10 days. Subsequently, colony formation was analyzed. *P<0.05 and **P<0.01 vs. vehicle. 5-FU, 5-fluorouracil; CCK8, Cell Counting Kit-8; Ctrl, empty vector; PLEK2-OE, pleckstrin 2 overexpressing; si/siRNA, small interfering RNA; siCtrl, negative control siRNA.
Supplier Page from Abcam for Anti-PLEK2 antibody