Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

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

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 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/.

PMC Copyright notice

Fig. 7 — A IGF1R-targeting mABs A12 (cixutumumab) and Tepro (teprotumumab) were expressed and purified using a GMP-like method and then conjugated to protamine to enable siRNA binding and transport. IgG-protamine conjugates exhibit a decent molecular weight shift (arrows). HC heavy chain, LC light chain, -P SMCC-protamine. B Band-shift assay using αIGF1R-mABs-protamine (-P) and different ratios of siRNA (left panels). Antibody-protamine complexes without free SMCC-protamine do not bind siRNA anymore. Right panels (a–d): αIGF1R-mAB-P in presence of free SMCC-protamine can form nanoparticles with Alexa488-control-siRNA (a: A12, b: Tepro). αIGF1R-mAB-P depleted from free SMCC-protamine cannot form these nanoparticles with Alexa488-control-siRNA (c: A12, d: Tepro). C Anti-IGF1R-mAB-protamine with free SMCC-protamine shuttled Alexa488-marked control-siRNA to SK-N-MC Ewing cells (left panel: A12-mAB-P, right panel: Tepro-mAB-P). Bar represents 10 µm. D Schematic representation of the breakpoint regions in the human EWS protein, the FLI-1 protein and the resulting oncogenic fusion protein EWS-FLI1 along with the breakpoint mRNA sequence and the EWS-FLI1-specific siRNA sequence that was used for this study (see E–G and Fig. 8) (BD binding domain, NLS nuclear localisation signal). E–G SK-N-MC cells were treated with protamine-conjugated A12 (E) or Tepro (F) formed siRNA nanoparticles as indicated and subjected to colony formation assays. E/F–siRNA is an siRNA interfering with the mRNA of the driving Ewing sarcoma EWS-FLI1 as depicted in D. G SK-N-MC cells treated with αIGF1R (A12)-mAB-protamine/EWS-FLI1-siRNA/P nanoparticles form significantly less colonies in soft agar than cells treated with αIGF1R (A12)-mAB-protamine/contr (scr)-siRNA/P. No differences in colony formation can be observed when SK-N-MC cells were treated with αIGF1R-mAB-protamine conjugates without free SMCC-protamine not resulting in the formation of nanocarriers. Shown here are means of three independent experiments ± SD. Asterisk indicates significant differences (p < 0.05, two-sided t-test).