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. 2022 Feb 26;41(15):2210–2224. doi: 10.1038/s41388-022-02241-w

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© The Author(s) 2022

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 — A Coomassie-stained SDS-PAGE showing αEGFR-mAB-P, αEGFR-mAB-P coupled with 32x SMCC-protamine and HPLC-fractions 25–31 of anti-EGFR-mAB coupled with 32x SMCC-protamine after depletion of unbound SMCC-protamine; HC heavy chain, LC light chain, -P SMCC-protamine. B Band-shift assays showing that non-SMCC-protamine-depleted αEGFR-mAB-P binds siRNA in a 1:8 to 1:16 molar ratio (left part), whereas the chromatographically depleted αEGFR-mAB-P does not bind siRNA. C–E Dynamics of internalisation of Alexa488-siRNA by confocal internalisation studies with depleted vs. non-depleted αEGFR-mAB-P with fluorescence-tagged siRNA on A549 NSCLC cells. Blue fluorescence for nuclei, red for actin, green for internalised Alexa488-siRNA. C Non-depleted αEGFR-mAB-P transports Alexa488-siRNA to A549 intracellular vesicles. Please note the cytoplasmic and perinuclear localisation of Alexa488-positive vesicles (compare also to Supplementary Fig. 5). The purification process leads to abolished internalisation of siRNA (D). SMCC-protamine alone does not work as an unspecific transfection agent (E). Scale bars 10 µm. F Dynamics of Alexa488-siRNA internalisation to A549 cells mediated by non-depleted αEGFR-mAB-P/P vs. SMCC-protamine-depleted αEGFR-mAB-P, controlled by SMCC-protamine only as carrier molecule in flow cytometric analysis (n = 3; error bars indicate SEM). Statistical significance was tested by two-way ANOVA with subsequent Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Of note, only the non-SMCC-protamine-depleted conjugate presented a significant siRNA internalisation compared to SMCC-protamine-depleted and SMCC-protamine from 6 h into A549 cells. G, H Functional relevance of free SMCC-protamine in the reaction mixture. Non-depleted αEGFR-mAB-P-transported KRAS siRNA leads to significant reduction of colony formation in NSCLC A549 cells (G) and NSCLC SK-LU1 cells (H) in comparison to control (scrambled, scr) siRNA, whereas the depletion of free SMCC-protamine completely abolishes this effect. SMCC-protamine alone (without αEGFR-mAB) again is not able to efficiently transport and internalise KRAS siRNA seen by no effect in colony assay (H, right bars; Mean ± SD of three independent experiments. Two-sided t-test, *p < 0.05).