Supporting Figures

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Fig. 5. Down-regulation of FADD-like IL-1b-converting enzyme (FLICE)-associated huge protein (FLASH) results in S-phase block. Shown is cell cycle distribution of SAOS2 (a), HEK293 (b), MCF-7 (c), and 3T3 (d) cells and MEFs (e) transfected with GFP-spectrin and either pSUPER-scrambled (Left, control) or pSUPER-FLASH-1 (Right, shFLASH). Cells were stained with PI, and GFP-positive cells were analyzed by flow cytometry for DNA content. Percentages of cells in G₁, S, or G₂/M, calculated by using ModFit program, are indicated. Identical results were obtained by using pSUPER-FLASH-2.

Fig. 6. FLASH colocalizes with p220/NPAT. Coimmunostaining using anti-FLASH (green) and anti-NPAT (red) antibodies in primary human fibroblasts (hFibr) and coimmunostaining using anti-FLASH (red) and anti-NPAT (green) antibodies in HeLa, IMR90, and HaCat. In all cells, the endogenous proteins colocalize.

Fig. 7. Down-regulation of FLASH does not affect general transcription. HeLa cells transfected with GFP-spectrin together with either pSUPER-FLASH-1 or pSUPER-scrambled were incubated 48 h after transfection with 1 mM BrU for 30 min. Cells were harvested and fixed in cold 1% paraformaldehyde for 15 min and permeabilized with 70% ethanol at -20°C. Cells were then subjected to anti-BrU immunofluorescence, and GFP positive cells were analyzed by flow cytometry. (Left) A representative experiment. pSUPER-scrambled cells are in gray, and pSUPER-FLASH-1 cells are in red; no difference in BrU incorporation is observed. In one of four experiments performed, a modest but not statistically significant increase of BrU incorporation was observed in cells transfected with pSUPER-FLASH-1 compared with cells transfected with pSUPER-scrambled. (Right) A Kolmogorov-Smirnov statistical analysis showing the perfect overlap of the two curves.