FIGURE 5.

hCLE controls RNA translation. HEK293T cells were infected with lentivirus expressing a control siRNA (siCt) or a siRNA that targets hCLE (siCLE). (A) Western blot detection of hCLE in control or silenced cells. (B) HEK293T cells were infected with control (siCt) or a siRNA that targets hCLE lentiviruses. Control or silenced HEK293T cells were cultured in Met/Cys-depleted DMEM (30 min); cells were untreated (DMSO) or actinomycin D (Act D)-treated (30 min), followed by ³⁵S-Met-Cys addition (time 0). At the indicated times aliquots were taken and used for SDS-gels and quantitated using ImageJ application (bottom). Rate represents the differences between signals obtained at 1h after ³⁵S-Met-Cys addition and those obtained at 5 h. A representative image is shown. The experiment was repeated three times. (C) HEK293T cells were infected with lentivirus expressing a control siRNA (siCt) or a siRNA that targets hCLE. Control or silenced HEK293T cells were cultured in Met/Cys-depleted DMEM (30 min) and then treated with 80 μM of RNAP III inhibitor followed by ³⁵S-Met-Cys addition (time 0). At the indicated times aliquots were taken and used for SDS-gels and quantitated using ImageJ application. A representative image is shown. (D) Quantitation of data obtained from part C. The experiment was repeated two times.