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. 2005 Dec;25(23):10492–10506. doi: 10.1128/MCB.25.23.10492-10506.2005

FIG. 3.

FIG. 3.

Accumulation of the DNA damage foci γH2AX and 53BP1 by acute withdrawal of WRN and BLM in WI-38 primary fibroblasts. (A) Primary WI-38 cells (PDL 26) at ∼30% confluence were transfected with the various siRNAs and cultured in the presence of 10% serum. At 4 days posttransfection, immunofluorescence analysis for γH2AX and 53BP1 foci was performed. The figure shows representative patterns of γH2AX and 53BP1 foci formation after WRN RNAi using anti-γH2AX and 53BP1 mouse MAbs (green and red, respectively). OF, Oligofectamine. (B) γH2AX foci accumulation by acute WRN and BLM depletion in the presence of the general caspase inhibitor Z-VAD-FMK. The cells were grown in the presence of 20 μM Z-VAD-FMK from the time of siRNA transfection as described in Fig. 2. Histograms indicate the percentage of cells with γH2AX foci in primary, asynchronous WI-38 cells after various siRNA transfections, as indicated. On average, more than 250 intact nuclei were screened per individual condition, and the data represent the means and SEMs of triplicate samples. (C) Generation of RNAi-resistant full-length WRN and BLM cDNAs by site-directed mutagenesis. The targeted sequences highlighted and the mutagenized nucleotides are indicated in boldface. (D) Specificity of the WRN and BLM RNAi-resistant full-length cDNA constructs. Real time RT-PCR was performed after sequential transfections. After 16 to 24 h of cell splitting, the cells were transfected with the various cDNAs, followed by siRNA transfection within 16 h, as indicated. Total RNA was isolated after 36 h of the siRNA transfections. (E) Complementation of the WRN siRNA-induced DNA damage response by RNAi-resistant full-length WRN gene. Sequential transfections were performed as described above. The cells were fixed 4 days after siRNA transfections, as indicated. The percentage of γH2AX-positive cells expressed as the fold changes compared to the value of the control cells treated the transfection reagent, Metafectene (M), alone. Error bars represent the SEMs of three experiments. Co-V, control expression plasmid; pWRN, RNAi-resistant full-length WRN cDNA; SCR, scrambled siRNA control.