Fig. 2.

SPIDR is required for BLM foci formation and suppresses SCE. (A) Quantitative RT-PCR showing SPIDR mRNA levels are down-regulated by siRNAs. Bars represent the average of three experiments, and error bars are SDs. (B) Representative immunoblotting of cells transfected with indicated siRNAs. (C and D) SPIDR is required for BLM foci formation. U2OS cells were transfected twice with control siRNA, or siRNAs specific for SPIDR or BLM. Forty-eight hours after transfection, cells were treated with HU (2 mM) for 16 h or CPT (1 μM) for 3 h before fixing and processed for BLM and γH2AX immunofluorescence. Representative BLM foci (HU treatment) are shown (C). (Magnification: 100×.) Quantification results were the average of three independent experiments and were presented as mean ± SEM (D). (E–G) Rescue of BLM foci formation by expression of a siRNA#1-resistant SPIDR cDNA. A U2OS cell line to express siRNA#1-resistant SPIDR (SFB-SPIDR-SiR) under the control of a tetracycline-inducible promoter was generated. The resulting cell line was transfected with indicated siRNAs and was either left uninduced (−) or was induced (+) by doxycycline (Dox) addition for 24 h before HU (2 mM) or CPT (1 μM) treatment. After 16 h of treatment with HU or 3 h of treatment with CPT, cells were fixed and processed for BLM immunofluorescence. The exogenous SPIDR expression was confirmed by immunoblotting using anti-Flag antibody (E). Representative BLM foci (HU treatment) were shown (F). (Magnification: 100×.) Quantification results were the average of three independent experiments and were presented as mean ± SEM (G). (H) SPIDR suppresses SCE. Representative metaphase spreads showing SCEs from HeLa cells transfected with control or SPIDR siRNAs (red arrows). (Magnification: 100×.)