Figure 3. FAVL elevation compromises the FA-BRCA pathway.

(A) FANCD2 activation is compromised in U2OS cells expressing Flag-FAVL. U2OS cells were transfected with various amounts of FAVL plasmid and treated with 40 ng/ml mitomycin C (MMC) 24 hours after transfection. The level of activated FANCD2 in these transfected cells (marked with the red arrow) decreased more when a higher amount of FAVL plasmid was transfected. The focus formation of FANCD2 is shown, derived from the same batch of cells transfected with 10 μg of FAVL cDNA–containing plasmid. The relevant controls and similar data obtained from the other cells are shown in Supplemental Figure 3A. (B) The FA-BRCA pathway is defective in 3 additional lung cancer cell lines, Hop62, UMC11, and HT182, expressing high levels of FAVL. Levels of monoubiquitinated FANCD2 were examined in all 17 lung cell lines following treatment with 25 ng/ml mitomycin C. Besides Calu-6 cells, FANCD2 activation was clearly compromised in Hop62, UMC11, and HT182 cells (data not shown) and compromised to some extent in A549, H650, H526, and H82 cells (Supplemental Figure 3B). FANCD2 activation in Hop62, UMC11, Calu-6, and HT182 cells remained defective after treatment with higher doses of mitomycin C (50 ng/ml and 100 ng/ml). Analysis of lysates from A549 cells, carrying an intact FA-BRCA pathway, is provided for comparison. FANCD2 foci were examined in mitomycin C–treated cells, at the dose of 100 ng/ml, and ubiquitinated FANCD2 was also examined by Western blotting in these corresponding cells (Supplemental Figure 3B). The L/S ratio in A549 cells (marked in red) is much higher than that in other cells (marked in black).