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. 2020 Aug 27;39(40):6370–6386. doi: 10.1038/s41388-020-01433-6

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© The Author(s), under exclusive licence to Springer Nature Limited 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. 4 — a Experimental timeline of cells studied with optical (OM) and transmission electron microscopy (TEM). MDAH and TOV21G cells were treated with GSK650394 (GSK) 35 µM for 36 h. b Representative optical microscopy pictures of MDAH and TOV21G cells treated or not with GSK650394 (GSK) as reported in a. Cytoplasmic vacuoles are present only in GSK650394 (GSK) treated cells. Scale bars = 50 µm. c TEM ultrastructure analyses of MDAH (left panels) and TOV21G (right panels) cells. a and e Untreated cells; b and f GSK650394-treated cells (GSK) showing cytoplasmic vacuoles accumulation; c and g Magnification of initial autophagic vacuoles (AVi) (white arrowheads indicate double-membrane) and degradative autophagic vacuoles (AVd) vacuoles (d and h). Highlighted intracellular organelles in untreated cells are endoplasmic reticulum (ER), Mitochondrion (m) and Golgi apparatus (Go). Scale bar: 300 nm. d Western blot analyses evaluating the expression of the autophagy markers LC3I/LC3II and p62 in MDAH and TOV21G cells, treated with GSK650394 35 μM (36 h), CBDCA 140 μg/ml (16 h) and GSK+CBDCA (as in Fig. 2d) compared to the effect of Bafilomycin A1 (Baf A1). Vinculin was used as loading control. (See also Figs. S5 and S6).