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. 2024 Dec 30;14:31838. doi: 10.1038/s41598-024-82957-9

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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-nc-nd/4.0/.

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Fig. 2 — AMBRA1 is required for CD8⁺ T cell differentiation to CTLs. (A) Immunoblot analysis of human AMBRA1 levels in control (ctr, scramble RNAi) and AMBRA1 KD CTLs (KD). Actin and ß-tubulin were used as loading controls. The migration of molecular mass markers is indicated. The histogram shows the quantification of AMBRA1 expression in CTLs normalized to actin or ß-tubulin (n = 3, one sample t test, ctr value = 1). (B) RT-qPCR analysis of human RUNX3, T-BET and EOMES mRNA in control and AMBRA1 KD CTLs. 18S was used for normalization (n_donor = 3, one sample t test, ctr value = 1). (C) RT-qPCR analysis of the GZMA, GZMB, PRF, GNLY and SRGN mRNA and (D) Immunoblot analysis of the LG components GZMB and PRF in control and AMBRA1 KD CTLs. Actin was used as loading control. The migration of molecular mass markers is indicated. The histogram shows the quantification of GZMB and PRF expression in AMBRA1 KD CTLs related to control CTLs (n = 3, one sample t test, ctr value = 1). (E) Real-time calcein release-based killing assay. Control or AMBRA1 KD CTLs were co-cultured with sAg-loaded Raji B cells at the target:effector (T:E) ratios indicated. The graph shows the kinetics of target cell killing quantified by measuring calcein fluorescence every 10 min for 4 h. The histogram shows the quantification of the percentage of target cell death at the endpoint (4 h) of independent experiments carried out on CTLs from n_donor = 3, performed in duplicate (one-way ANOVA test). Data are shown as mean fold ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.