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. 2020 Jan 27;11(1):69. doi: 10.1038/s41419-020-2266-x

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© The Author(s) 2020

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/.

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Fig. 1 — Immunoblot analysis of the histone modifying enzyme SETD2 (a) and its histone target H3K36me3 (b) expression levels in ACHN and CAKI-1 renal clear carcinoma cells confirmed the SETD2 deficiency in the last named RCC cell line. c Immunoblot analysis of LC3 shows decreased expression level in SETD2-deficient CAKI-1 cells as compared with SETD2-wild-type ACHN cells. Treatment with Bafilomycin A1 (BafA1, 40 nM) for 4 h shows a significant decrease in LC3-II lipidation in CAKI-1 cells as compared with ACHN cells. d Quantification of LC3-II expression compared with the expression of the housekeeping gene, actin, with and without BafA1 treatment in both cell lines. e Quantification of LC3-II/LC3-I ratio to monitor autophagic flux in ACHN and CAKI-1 cells with and without BafA1 treatment demonstrates that cells that lacks SETD2 expression exhibit a decreased autophagic flux. f Analysis of MAP1LC3B mRNA expression by qPCR in SETD2-deficient CAKI-1 cells and SETD2-competent ACHN cells. Bars display the mean of four (d, e) or three (f) experiments, error bars represent SEM; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05.