Fig 1.
Effect of PKM2 silencing on OXA cytotoxicity in HT29, SW480 and HCT116 cells.
Dose-response curves for HT29, SW480 and HCT116 cell lines after PKM2 gene silencing and OXA treatment at 0–140 μM and 0–32 μM for 24 hours. Curves represent the average values from at least three independent experiments. Cell proliferation was measured by MTT assay. Vertical bars in the graphics represent ± SD. Insets show PKM2 inmunoblotting after siRNA-directed inhibition. Specific IC50 values for oxaliplatin in all conditions are displayed in the table. IC50 values for Mock conditions (without transfection) were very similar to those of siNTC and are not shown. *p-values are result of comparison to the siNTC condition.
Fig 2.
Effect of PKM2 silencing according to p53 in HT29 and HCT116 cell lines.
Bars represent the IC50 ± SD (average values from at least three independent experiments) for oxaliplatin for each condition. Insets show PKM2 and p53 inmunoblotting after PKM2 gene silencing. Specific IC50 values for oxaliplatin in all conditions are displayed in the table. IC50 values for Mock conditions (without transfection) were very similar to those of siNTC and are not shown. *p-values are result of comparison to the siNTC condition. * P- value < 0.05. ** P-value < 0.01; *** P-value < 0.001
Fig 3.
PKM2 silencing alters OXA response in HT29 cells but not apoptosis activation.
After siRNA transfection cells were treated with 15 μM OXA for 24 h period, observed by optical microscopy at 0, 24 and 48 h after the end of drug exposure (0 h refers to cells treated for 24h; 24 h refers to cells treated for 24 h and left to recover for additional 24 h) (A) and quantified by trypan blue staining (B). Apoptosis activation after 24 and 48 hours of 10 μM OXA exposure was determined by FITC-Annexin V/ PI double staining (C) and measured as a ratio between percentages of apoptotic treated (T) and non-treated (NT) cells (D). Vertical bars in the graphics represent ± SD. *P-value < 0.05. NT: non-treated cells. Optical microscopy: objective 10x magnification.
Fig 4.
Cell cycle distribution in HT29 and HCT116 cells after PKM2 gene silencing and treatment with OXA.
Both cell lines were transfected and/or exposed to 10 μM OXA for 8, 24, 48 and 72 h. After propidium iodide staining, the proportion of cells in cell cycle phases G1, S and G2/M was measured by flow cytometry and quantified by Flowjo v9.2 software. Results are representative of at least three independent experiments. P-values ≤ 0.05 are represented as a *.
Fig 5.
PKM2 subcellular localization in response to OXA in colorectal cancer cell lines.
Immunoflourescence staining of PKM2 (red) demonstrates nuclear accumulation in HCT116 and HT29 cells after treatment with OXA in a time and dose-dependent manner but not in resistant HTOXAR3 cells. Nuclei were stained in blue. NT: Non-treated cells. Objective lens: 40x immersion oil. Scale bar: 10 μm.
Fig 6.
Changes in cell death genes expression patterns after PKM2 gene silencing and/or OXA treatment.
A. 3-D plot showing fold changes in expression patterns after treatment with 10 μM OXA in HT29, siPKM2-HT29 and HTOXAR3 cells. B. Heat map showing up- and down-regulated genes after OXA treatment according to three different cell death pathways.
Table 1.
Most relevant changes in cell death genes expression patterns after treatment with OXA and/or PKM2 gene silencing.
Fig 7.
BMF is involved in oxaliplatin response and cell death at the transcriptional level A.
Changes in BMF gene expression between OXA treated (T) and non-treated (NT) HT29, siPKM2- HT29 and HTOXAR3 cells. Vertical bars in the graphics represent means obtained from at least 3 independent experiments ± SD. B. Percentage of dead cells after treatment with oxaliplatin and/or BMF gene knockdown. Bars represent means obtained from at least 3 independent experiments ± SD. The Little graph shows percentage (mean ± SD) of BMF expression inhibition after siRNA transfections. * P- value < 0.05. ** P-value < 0.01; *** P-value < 0.001