Extended Data Fig. 6: Employing tuned SMART-SpyCatchers.
a, SpyCatcher003 utilizes its residue K31 to form an isopeptide bond with D117 of SpyTag003 upon binding. A mutant of SpyN carrying K31E was evaluated for its abilty to recruit SpyTag003-AF594 upon SMART actuation of SpyCatcher003. The data represent the flow cytometry analysis of K562HER2+/EGFR+ cells left untreated or treated with either SMART-SpyCatcher or SMART-SpyCatcherK31E (both at 100 nM, employing eNrdJ-1cage and operating through [HER2 AND EGFR] gating). RFU denotes relative fluorescence units. The relative mean AF594 MFI is given in the bar graph on the right with error bars signifying the standard error mean (n = 3 independent biological replicates) and statistical significance evaluated using an unpaired two-sided t-test (ns = not significant; ****P < 0.0001). b, The total cellular levels of HER2 and EpCAM for MCF-10a, MCF-7, and Sk-br-3 cell lines were determined by Western blotting (left) and quantified (right). Intensity values were corrected against the GAPDH signal for each cell line and further normalized to the overall highest level (Sk-br-3 for HER2, MCF-7 for EpCAM). c, The endogenous surface levels of HER2 and EpCAM on mammary cell lines MCF-10a, MCF-7, and Sk-br-3 were furthermore profiled and categorized as low (MFI < 1000) or high (MFI ≥ 1000). Errors = standard error mean (n = 3 independent biological replicates). d, MCF-10a cells either unstained or stained with the cell-permeable dye 5-Chloromethylfluorescein diacetate (CMFDA) were also phenotyped to determine any differences in the levels of surface HER2 and EpCAM. Errors = standard error mean (n = 3 independent biological replicates). Statistical significance was evaluated using an unpaired two-sided t-test (ns = not significant). e, SMART-SpyCatcher assigned for [HER2 AND EpCAM] logic was tested in flow cytometry experiments using mixed mammary cell population 1 (equal amounts of MCF-10aHER2low/EpCAMlow and MCF-7HER2low/EpCAMhigh) and mixed mammary population 2 (equal amounts of MCF-10aHER2low/EpCAMlow and Sk-br-3HER2high/EpCAMhigh). MCF-10a cells were pre-stained with CMFDA, which labels intracellular proteins. Each population was incubated with 100 nM SpyTag003-AF594 in the absence or presence of 100 nM αHER2-SpyN and SpyC-αEpCAM (employing eNrdJ-1cage with K114AK116A). Shown are representative flow cytometry plots of the recruitment of SpyTag-AF594 by the subpopulations under the different conditions. f, SMART-SpyCatcher003 using different versions of eNrdJ-1cage were tested on the cell lines (color coded as in panel e) and the quantified data presents the mean of the individual median fluorescence intensities (MFI) of the AF594 signal with error bars signifying the standard error mean (n = 3 independent biological replicates). Statistical analysis using paired two-sided t-test.
