Efficient knockdown of FA proteins in hESCs by RNAi. (A) Schematic of the lentiviral vector used to deliver small interfering RNAs. (B) FANCD2 Western blot on lysates from GFP⁺ FACS for hESCs infected at MOI 10 and 50 with empty vector, LUCi, and FANCD2i virus at day 4 after infection. Percentage of knockdown is calculated using ImageJ software (National Institutes of Health) after normalization to α-tubulin levels. (C-D) FANCA Western blot on lysates from GFP⁺ FACS of hESCs infected either with empty vector, LUCi, and FANCAi virus at MOI 50 (C) or increasing MOIs of FANCAi virus (D) at day 4 after infection. Percentage of knockdown is calculated using ImageJ software after normalization to a nonspecific band. (D lane 5, FA-A) A lysate from a FA-A patient fibroblast; (lane 6, +A) the patient fibroblast line retrovirally corrected with FANCA.