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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. 3 — The AMBRA1 activator ULK1 is required for CTL differentiation and function. (A) RT-qPCR analysis of human RUNX3 and T-BET mRNA in CTLs treated with SBI-0206965 or vehicle (DMSO).18S was used for normalization. Data are shown as mean fold ± SD of treated vs untreated CTLs (n_donor = 3, one sample t test, vehicle value in CTLs set as 1). (B) RT-qPCR analysis of the GZMA, GZMB, PRF, GNLY and SRGN mRNA in CTLs treated with SBI-0206965 or vehicle. Data are shown as mean fold ± SD of treated vs untreated CTLs. 18S was used for normalization (n_donor = 3, one sample t test, vehicle value = 1). (C) Immunoblot analysis of the LG components GZMB and PRF in CTLs treated with SBI-0206965 or vehicle. Actin was used as loading control. The migration of molecular mass markers is indicated. The histogram shows the quantification of GZMB and PRF protein expression in SBI-0206965-treated CTLs related to untreated CTLs. Data are shown as mean fold ± SD (n_donor = 3, one sample t test, vehicle value = 1). (D) Real-time calcein release-based killing assay. CTLs either untreated or treated with SBI-0206965 were co-cultured with sAg-loaded Raji B cells at the target:effector 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. Data are shown as mean fold ± SD (n = 3, one-way ANOVA test). ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.