Fig. 2.

Knockdown of endogenous CKMT1 results in apoptosis. (A) Semi-quantitative RT-PCR at 48 h and 72 h post siRNA transfection using primers specific for human CKMT1 and β-actin in HeLa cells. (B) Western blot analysis for CKMT1 expression. HeLa cells were transfected with siRNA against CKMT1 (siCK1) or with a scrambled control (sc) sequence. PARP cleavage and activation of caspase 3 were observed after siRNA transfection in HeLa cells. The antibody against caspase 3 recognises only the cleaved product. (C) PI staining and subsequent subG1-G0 analysis by FACS. Representative FACS profiles and subG1-G0 gating are shown on the right. Data show the mean±s.d. of three independent experiments. (D) PI and annexin V co-staining and subsequent FACS analysis post transfection with siCK1. Representative FACS density blots are shown, with the percentages representing the means of three independent experiments. (E) Quantification of caspase 3 and Bax activation by immunostaining and subsequent FACS analysis using Alexa-Fluor-488- and Alexa-Fluor-633-conjugated secondary antibodies monitored over 4 days post transfection. (F) Inhibition of the induction of cell death that is mediated by siCK1 by treatment of cells with the pan-caspase inhibitor. HeLa cells were transfected with siCK1 or sc, and treated with the indicated concentration of zVAD at 24 h and 48 h post transfection. Cell death was assessed by PI exclusion staining and subsequent FACS analysis. Data show the mean±s.d. of three independent experiments. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test).