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. 2011 May 24;6(5):e20276. doi: 10.1371/journal.pone.0020276

Figure 4. Increased 5-FU resistance in CALB2 silenced cells is associated with abrogated induction of the intrinsic apoptotic pathway.

Figure 4

(A) Western blot analysis of CALB2 in the mitochondrial and cytosolic fractions of p53+/+ HCT116 cells following transfection with control siRNA (−) or CALB2-targeting siRNA (+) and 72 h co-treatment with 5-FU. Cox IV was used as a mitochondrial loading control and α-tubulin was used as a cytosolic loading control. (B) TMRE flow cytometric analysis showing the percentage of isogenic p53+/+ and p53−/− HCT116 cells with loss of Δψm (% depolarised) following transfection with control siRNA (sc) or CALB2 targeting siRNA (siCALB2) and 72 h co-treatment with 5 µM 5-FU. **p<0.01, error bars represent mean ± SEM. (C) TMRE flow cytometry experiment repeated with a second siCALB2 sequence. (D) Western blot analysis of smac and cytochrome C in the mitochondrial and cytosolic fractions of 5-FU treated p53+/+ HCT116 cells. Cox IV and α-tubulin were used as loading controls. (E) Caspase activity assays in p53+/+ HCT116 cells. RFU values were normalised to cell viability (MTT assay) and expressed as fold-change RFU in the CALB2 silenced cells relative to the siRNA control (SC) cells. Error bars represent the mean ± SEM. (F) Annexin V/propidium iodide flow cytometric analysis of apoptotic p53+/+ HCT116 cells following CALB2 or XIAP silencing alone or in combination. Cells were co-treated with 5 µM 5-FU for 72 h as indicated. *p<0.05, **p<0.01, error bars represent mean ± SEM. (Inset) Western blot analysis of XIAP expression following transfection with 10 nM control siRNA (−) or 10 nM XIAP targeted siRNA (+). GAPDH was used as a loading control.