Overexpression of FANCF in 8226/S and U266/S reduced melphalan response, whereas silencing FANCF in 8226/LR5 and U266/LR6 enhanced melphalan response, respectively. (Ai) MTT assay. Overexpression of FANCF in drug-sensitive 8226/S and U266/S cells enhanced cell survival. The data are presented as percent survival above control cells. The experiment was repeated at least 3 times. Representative results are shown. (ii) IC50 is the mean of 3 independent experiments and SD. Student t test was used for statistical analysis. ^*P < .05. The experiments were repeated 3 times. (iii) Empty vector-transfected 8226-PQC and U266-PQC cells served as controls for FANCF-transfected 8226-FANCF and U266-PQC cells. β-Actin blot served as loading control. (Bi) Apoptosis assay. Transfection of siFANCF partially reversed melphalan resistance in 8226/LR5 and U266/LR6 cells. Annexin-V-FITC staining was used. The percentage of annexin-V-FITC⁺ cells is labeled, and specific apoptosis has been calculated. The mean values and standard deviations from a representative experiment performed in triplicate are shown. Student t test was used for statistical analysis. ^*P < .05. (Biii) Western blot analysis of FANCD2 and FANCF showed reduced protein expression in 8226/LR5 and U266/LR6 cells transfected with siFANCF compared with cells transfected with siLuc as a control. β-Actin blot served as loading control. (C) Capacity to repair ICLs was reduced in FANCF knockdown LR5 and LR6 cells compared with control siLuc-transfected cells, LR5 siLuc (top) and LR6 siLuc (bottom), respectively. The mean values and standard deviations from 3 independent experiments are shown. Student t test was used for statistical analysis. ^*P < .05.