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. 2024 Oct 9;15:8731. doi: 10.1038/s41467-024-52975-2

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 6 — a Experimental design to quantitate fluorescent soluble cargo uptake in cells pre-treated with cargo-saturated CYpHER. Step 1: CYpHER is saturated with target (2:1 target:binding moiety ratio), then applied to cells for 2 h. Step 2: After cells are thoroughly rinsed, new fluorescently-labeled soluble target is added to cells. Fluorescence accumulation is increased by CYpHER pre-treatment. b Designs and elements of CYpHERs used. c Soluble EGFRvIII uptake per cell after 24 h incubation with A549, H1650, H1975, or H358 cells either untreated (to quantitate passive uptake) or pre-treated for 2 h with unlabeled-EGFR-saturated 10 nM CT-1212-1, normalized to each cell line’s untreated uptake levels. N cells per sample as follows. H358: No CYpHER, 6309; CT-1212-1 Pre-treat, 5901. H1650: No CYpHER, 6622; CT-1212-1 Pre-treat, 6567. H1975: No CYpHER, 6087; CT-1212-1 Pre-treat, 5858. A549: No CYpHER, 7584; CT-1212-1 Pre-treat, 7848. Each line (H358, H1650, H1975, A549), pre-treatment vs Untreated via two-tailed Kolmogorov–Smirnov [KS] test were P < 0.0001. Pre-treatments all-vs-all by Kruskal–Wallis test with Dunn’s correction [KWD] were P < 0.0001. d Soluble EGFRvIII uptake per cell in H1975 cells as in c comparing CT-1212-1, CT-6212-1, and CT-5212-3. N cells per sample: CT-1212-1 Pre-treat, 5778; CT-6212-1 Pre-treat, 5277; CT-5212-3 Pre-treat, 5540. All-vs-all by KWD: CT-6212-1 Pre-treat vs CT-5212-3 Pre-treat P > 0.9999, all others P < 0.0001. e Soluble PD-L1 uptake per cell as in c except with soluble PD-L1 as cargo, comparing CT-4212-1 and CT-4212-3 in MDA-MB-231 (left) or H1650 (right) cells. N cells per sample as follows. MDA-MB-231: No CYpHER, 22023; CT-4212-1 Pre-treat, 6667; CT-4212-3 Pre-treat, 7398. H1650: No CYpHER, 20807; CT-4212-1 Pre-treat, 7546; CT-4212-3 Pre-treat, 7215. Within each cell line, all-vs-all KWD were P < 0.0001. Each experiment in c–e was performed once. All box plots (c–e) feature a median (black line), 25^th and 75^th percentiles (box boundaries), and 5^th and 95^th percentiles (whiskers). See the Supplementary Data for full statistical breakdown. Source data are provided as a Source Data file.