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. 2018 May 8;13(5):e0197029. doi: 10.1371/journal.pone.0197029

Fig 6. Engineered Fn3 protein variants 1.4.1 and 2.4.1 localized to early endosomes upon binding MSLN.

Fig 6

Analysis by (A) flow cytometry and (B) imaging flow cytometry confirms MSLN presence on the surface of KB-3-1 cells compared to the MSLN-negative MCF-7 cells, as detected by an anti-MSLN antibody. (C, D) KB-3-1 cells (top) internalize AF488-labeled 1.4.1 (C) and AF488-labeled 2.4.1 (D), while MCF-7 cells show no specific binding or internalization (C bottom, D bottom). Endosomes are detected by an AF647-conjuated antibody recognizing the EAA1 early endosomal marker. Yellow in the merged images indicate co-localization between AF488-1.4.1 or AF488-2.4.1 anti-MSLN engineered proteins (green) and EEA1 (red). Original magnification 40X. Co-localization is quantified by the Bright Detail Similarity (BDS) metric, with values near 1 indicating co-localization. KB-3-1 BDS = 1.31 and 0.919 for AF488-1.4.1 and AF488-2.4.1, respectively. BDS values are not quantifiable for the negative control cell line, due to insufficient fraction of negative control cell population staining for binding or internalization of engineered protein variants.