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. 2019 Oct 11;10:4635. doi: 10.1038/s41467-019-12554-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — NLC-cell interaction studies. a Concentration-dependent uptake of FAM-CGY assemblies by hCMEC/D3 cells at 24 h, 37 °C. The right panel shows a typical fluorescence microscopy and differential interference contrast (DIC) images of a live hCMEC/D3 cell post FAM-CGY (5 µM) challenge. b The effect of internalisation inhibitors on the uptake of FAM-CGY NLC (final concentration 5 µM) and Texas Red-labelled transferrin (5 µg per well) by hCMEC/D3 cells. The results represent mean fluorescent intensity with respect to Control (no metabolic inhibitor). Open column = transferrin; grey column = FAM-CGY. c FAM-CGY NLC (5 µM) uptake by hCMEC/D3 cells is competitively inhibited by partially iron saturated human transferrin (Tf) and a commercially propriety RAGE peptide (RAGE P). Test column = reference FAM-CGY NLC uptake (no inhibitors) with auto-fluorescence subtracted. Other incubations are compared with Test incubation. Bovine serum albumin (BSA) was used as an irrelevant protein. d Down-regulation of TfR in hCMEC/D3 cells with TfR-specific siRNA reduces Tf and FAM-CGY uptake. In the right panel, open column = control siRNA treated cells and grey column = TfR siRNA treated cells. e Comparison of TfR expression in MCF-7 and MCF-10A cells and FAM-CGY uptake. In the right panel, open column = MCF-7 cell line and grey column = MCF-10A cell line. f Diminished NLC (final concentration 5 μM) binding to MCF-7 cells at 4 °C on saturation of TfR binding sites. au = arbitrary unit. g The effect of RAGE downregulation in hCMEC/D3 with a RAGE-specific siRNA on TAMRA-labelled Aβ oligomer and FAM-CGY uptake. In the right panel, open column = control siRNA treated cells and grey column = RAGE siRNA treated cells. All incubations were done in triplicate and each experiment was repeated three times. Panels represent the mean value of three separate experiments ± s.d. and each dot indicates the mean of three technical replicates. *p < 0.05 and **p < 0.01, non-paired two-sided student t-test compared with respective controls. Source data are available in the Source Data file