Fig 2: Effect of the overexpression of Plk2 on chemosensitivity and apoptosis of CRC cells. Dose-response of chemotherapeutic agents on the growth effect of Plk2 and vector control CRC cells. Colo-678 cells were treated with DMSO or different concentration of (A) oxaliplatin or (B) 5-Fu for 72 h. HT-55 cells were treated with DMSO or different concentration of (C) oxaliplatin. The viable cell number was determined using an MTT assay. Data are plotted as the mean ± standard deviation of three independent experiments with six repeats for each experiment. (D) Western blot indicating the overexpression of Plk2, with GAPDH as endogenous control. Effects of the overexpression of Plk2 on apoptosis were examined. Cells infected with lentiviruses encoding Plk2 or vector control were treated with oxaliplatin (1 μM) or DMSO for 16 h. (E) Cell lysates were subjected to western blot analysis for cleaved PARP. (F) Cells were stained with Annexin V and PI, and analyzed using flow cytometry. (G) Apoptotic cell rates are shown as a bar plot. Data are representative of three independent experiments. The P-value was calculated using Student's t-test (unpaired). *P<0.05; **P<0.01. CRC, colorectal carcinoma; Plk2, polo-like kinase 2; PARP, poly(ADP-ribose) polymerase 1; shRNA; 5-Fu, fluorouracil; DMSO, dimethyl sulfoxide; PI, propidium iodide; IC50, half maximal inhibitory concentration.

Fig 3: The mitochondria-mediated apoptotic cell death via streptozotocin (STZ) in beta cells is inhibited by natural COA water. (A) Beta TC-6 islet cells were cultured in a medium prepared with general water (Control) or natural COA water and then treated with streptozotocin for 72 h. Cell lysates were analyzed by immunoblotting with the antibodies indicated. (B) Beta TC-6 cells were cultured in a medium prepared with general water (Control) or natural COA water and then treated by H2O2 (10 μM) for 24 h. Cell lysates were analyzed by immunoblotting with the antibodies indicated. (C) Pancreatic Beta TC-6 islet cells were cultured with indicated waters and then treated by Streptozotocin or NAC (1 mM). Cell lysates were analyzed by immunoblotting with the antibodies indicated. (D) Beta TC-6 cells were cultured in a medium prepared with general water or natural COA water and then treated with streptozotocin for 72 h. And some of these cells were treated with MG132 (10 μM, for 4 h) in a medium prepared with natural COA water. Cell lysates were analyzed by immunoblotting with the antibodies indicated. (E, F) Beta TC-6 cells were cultured in a medium prepared with general water (Control) or natural COA water and then treated with streptozotocin for 18 h. The cells were stained with MitoSOX (5 μM) for 30 min and then observed by fluorescence microscopy. Representative images of mitochondrial ROS in the Beta TC-6 cells (E). Quantification of cleaved Caspase-9, cleaved Caspase-3, cleaved Caspase-8, Plk1, Plk2, and Plk3 levels relative to β-actin levels. The signal of the band was quantified with Image J software. cCas-9, Cleaved Caspase-9; cCas-3, Cleaved Caspase-3; cCas-8, Cleaved Caspase-8. Statistical comparison of scatter plot and bar graph was performed by repeated measure ANOVA with multiple comparisons test; *P < 0.05, **P < 0.01, n.s., non-specific. All experiments were repeated independently at least three times with similar results.

Fig 4: Effect of Plk2 knockdown on chemosensitivity and apoptosis of CRC cells. Dose-response of chemotherapeautic agents on the growth effect of CRC cells transfected with Plk2-shRNA and scrambled control. Colo-6678 cells were treated with DMSO or different concentrations of (A) oxaliplatin or (B) 5-Fu for 72 h. HT-55 cells were treated with different concentrations of (C) oxaliplatin. The viable cell number was determined using an MTT assay. Data are plotted as the mean ± standard deviation of three independent experiments with six repeats for each experiment. (D) Western blot analysis indicated the knocked down expression of Plk2, with GAPDH as an endogenous control. Effect of Plk2 knockdown on apoptosis. Cells infected with lentiviruses encoding Plk2-targeting or scrambled shRNAs were treated with oxaliplatin (4 μM) or DMSO for 16 h. (E) Cell lysates were subjected to western blot analysis for cleaved PARP. (F) Cells were stained with Annexin V and PI and analyzed using flow cytometry. (G) Apoptotic cell rates are shown as a bar plot. Data are representative of three independent experiments. The P-value was calculated using Student's t-test (unpaired). *P<0.05; **P<0.01. CRC, colorectal carcinoma; Plk2, polo-like kinase 2; PARP, poly(ADP-ribose) polymerase 1; shRNA, short hairpin RNA; 5-Fu, fluorouracil; DMSO, dimethyl sulfoxide; PI, propidiumiodide; IC50, half maximal inhibitory concentration.

Fig 5: Correlation between the expression of Plk2 and chemosensitivity in CRC cell lines. (A) Gene expression of Plk2 in four CRC cell lines by RT-qPCR analysis. Relative expression (FC) of Plk2was calculated with GAPDH as a reference gene, and normalized to that of SW620 cells. Data are presented as the mean ± standard deviation from three independent experiments, with triplicate amplifications for each experiment. The overall difference in the expression of Plk2 among the four cell lines was calculated using one-way analysis variance (P=2.82×10−5). Multiple comparisons were calculated by Tukey's 'Honest Significant Difference' method. The P-value of multiple comparisons between SW620 and other three cells is shown. (B) Western blot analysis of protein levels of Plk2 in the four CRC cell lines. GAPDH shows equal sample loading. Dose-response to (C) oxaliplatin and (D) 5-Fu in the four CRC cell lines. Cells were treated with DMSO or different concentrations of chemotherapeutic drugs for 72 h. The viable cell number was determined by cell viability assay. Data is plotted as the mean ± standard deviation of three independent experiments with six repeats for each experiment. (E) Gene expression of Plk2 in four CRC cell lines, determined by a microarray from the CCLE database. Relative expression (FC) of Plk2 was normalized to that of SW620 cells. (F) Pharmacological data of three CRC cells from CCLE database. IC50 values of three chemotherapeutic drugs for different CRC cell lines are presented. Plk2, polo-like kinase 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CRC, colorectal carcinoma; FC, fold change; CCLE, Cancer Cell Line Encyclopedia; IC50, half maximal inhibitory concentration.