Figure 2 - Molecular target identification based on MAGIC technology.

(a) Schematic of the retroviral vector constructs used to make an expression library. The cDNAs prepared from BJ fibroblasts with TRF2^BM-induced senescence were fused to the 5' or 3' end of the EGFP gene of pMAGIC-N or pMAGIC-C vector, respectively. The mRFP translated from an IRES served as an internal negative control for magnetic field–directed translocation. (b) Chemical structures of CGK733 and its biotinylated derivative. CGK733-biotin (2) was designed and synthesized on the basis of our studies on the structure-activity relationship of CGK733 (data not shown; see Supplementary Fig. 6 online). CGK733-biotin was used to coat MNP. (c,d) Visual screening based on MAGIC technology. HeLa cells were infected with the retroviral EGFP-fusion protein expression library described in a. These cells were incubated with MNPs coated with CGK733 and TAT-HA2 (2:1 ratio) in the presence of 100 M chloroquine for 12 h, and then the subcellular localization of EGFP was examined after application (first row), removal (second row) and reapplication (third row) of an external magnetic field ('MF'). To address potential false positives, mRFP, bicistronically coexpressed with EGFP-fusion protein, was simultaneously monitored. Live-cell confocal images were taken with the focal plane at the cellular basal surface ('Bottom') or with the pinhole size increased to collect whole cell images ('Whole'). Arrowheads indicate positive clones. RT-PCR and sequence analysis of mRNAs from these cells identified ATM. Scale bars, 100 m.