Extended Data Fig. 2: Confocal microscopy of K562 cell lines treated with SMART-SpyCatcher for [HER2 AND EGFR] logic.

a, K562^HER2+/EGFR+ cells were treated with αHER2-SpyN (100 nM) and SpyC-αEGFR (100 nM) for 2 hr, followed by SpyTag003 labeled with Alexa Fluor 594 (SpyTag003-AF594, 100 nM) for 20 min. SMART-SpyCatcher employed either NrdJ-1N(C76V)^cage/NrdJ-1C^cage(C76V) or NrdJ-1N(C76V)^{cage(K109EK119A)}/NrdJ-1C^{cage(C76VD66K)} (referred to as eNrdJ-1^cage). Following washing, the live cells were analyzed by confocal microscopy. Cell nuclei were stained with Hoechst, while HER2 and EGFR were tagged with eGFP and iRFP, respectively. Scale bar equals 20 μm. All subsequent panels apply similar reaction and analysis conditions as in panel a using the SMART-SpyCatcher (eNrdJ-1^cage) [HER2 AND EGFR] system. b, K562^HER2+, K562^EGFR+, and K562^HER2+/EGFR+ cells were treated and analyzed individually. Scale bars equal 10 µm. c, To determine the colocalization of HER2, EGFR and SpyTag003-AF594 the signal intensities of eGFP, iRFP, and AF594 derived from the cells in panel b were plotted (yellow dotted lines). d, A mixed population consisting of equal amounts of K562 (wildtype), K562^HER2+, K562^EGFR+, and K562^HER2+/EGFR+ cells were treated and imaged. Representative single cells of each cell line from the treated mixture and their associated fluorescence signals are shown on the right. Scale bar equals 20 μm. Data are representative of two independent experiments.