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. 2022 Jul 15;12:12078. doi: 10.1038/s41598-022-15244-0

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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 licence, and indicate if changes were made. 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/4.0/.

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Effects of HA-CPNs on Proliferation and Stemness of Primary GBM Patient- derived cell lines. (A) U-251 MG cells treated with indicated concentrations of HA-CPNs or vehicle control (HA-CPN vehicle) and subjected to trypan blue exclusion assay over a time course of 6 days; (B) Proliferation of heterogeneous (Het.), CD44 + and CD44- populations of U-251 MG cells, FACS- enriched for HA-CPNs (HA-CPN +) compared to HA-CPN- cells, assessed using trypan blue exclusion assay; (C–G) Patient lines, HF3035, HF2927 and HF2303, treated with 24 HA-CPNs/ cell and/or HA-CPN vehicle control and subjected to: a viability assay- average number of viable cells assessed over three cell lines tested, quantified as % of total population via trypan blue exclusion assay (C), a proliferation assay over the time course of 12 days- cell number assessed via trypan blue exclusion assay at the indicated time points (D), neurosphere formation assay, representative images of HF3035 and HF2927 (left); scale bar = 100 μm. Number of spheres formed is quantified per every 10⁴ cells seeded, three lines averaged (E), assessment of an average diameter of spheres formed; three lines pooled for analysis (F), qRT-PCR analysis of mRNA levels of the indicated markers of stemness/differentiation (G). Data shown as mean ± s.d, n = 3, *p < 0.05, **p < 0.01; Student’s t-test.