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Fig. 1. ß-actin mutation leads to a resistance of TNF-induced cell death in L929 cells. (A) The fused mRNA of neo and an endogenous gene in a TNF-resistant L929 clonal cell line was amplified by 3'-RACE. The junction sequence of the fused cDNA is shown, which reveals that the viral insertion occurred 5' to the coding region of the ß-actin gene. The amino acid sequence at the C terminus of neo is shown beneath the cDNA sequence. The sequence introduced by viral vectors is shown in lowercase. The number in parentheses indicates the position relative to the start codon of ß-actin. (B) ß-actin protein is reduced in ß-actin mutant cells (Actin^mut). Western blotting was performed using anti-ß-actin antibody. Equal loading of total cell lysates was determined by staining an identical SDS-PAGE with Coomassie Blue and a portion of the picture is shown. (C) Wild-type and Actin^mut cells were treated with TNF (100 ng/ml) for different periods of time and cell viability was assessed by propidium iodide (PI) exclusion. Results represent the means±s.e. (n=3). (D) Stable cell lines were generated from Actin^mut cells by transfection of ß-actin expression vector (Actin^mut+actin) or empty vector (Actin^mut+vector). The expression level of ß-actin was determined as in (B). (E) The sensitivity to TNF-induced cell death in Actin^mut, Actin^mut+actin and Actin^mut+vector was measured as in (C). (F) Parental wild-type L929 cells were treated with siRNA for ß-actin as described in Materials and Methods for different periods of time as indicated. The level of ß-actin protein was determined as in (B). (G) The viability of the RNAi-treated L929 cells was measured as in (C) after 48 hours TNF treatment.

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