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. 2022 Jan 27;12:1443. doi: 10.1038/s41598-022-05161-7

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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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Induction of autophagy and Integrated Stress Response in HEK293T subject to hypoxia. Cells were cultured and either kept in normoxic conditions (NOR) or exposed to hypoxia (HYP) for 16 h in DMEM EV-free serum media. (a) Small EV release. Quantitation of small EVs by NTA measurement of vesicles in the supernatant of the 10,000×g centrifugation of the conditioned media. Graph shows the amount of EVs corrected by total protein content in cellular lysates. Graphs are mean ± SEM of n = 3 experiments, each measured in triplicate. Statistical analysis: Two tailed t-test, where *indicates p < 0.05. (b, c) Analysis of autophagy and ER stress/integrated stress response (ISR) markers. Cellular lysates were obtained from cells following 16 h of NOR/HYP exposure. Samples were resolved in SDS-PAGE and immunoblotted with specific antibodies against phospho-p70S6K at the Thr389 residue, p62, or phospho-eIF2α with β-tubulin used as loading control. (b) Representative blot and (c) Quantification of all the immunoblots carried out in Image J (NIH). Graphs show the mean ± SEM of N = 3. Statistical analysis: Two tailed t-test, where ***indicates p < 0.001.