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. 2021 Sep 14;40:289. doi: 10.1186/s13046-021-02066-7

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© The Author(s) 2021

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 6 — Alcohol-induced ER stress in PCa cells. (A) Representative IF images of giantin (green) and ATF6 (red) in LNCaP cells: control and treated with 50 mM EtOH for 96 h; bars, 10 μm. (B) Quantification of the ratio of nuclear/cytoplasmic ATF6 IF in cells presented in A (N = 3; **P < 0.001, t test). (C) ATF6 W-B of the nuclear fraction from cells presented in A; lamin B1 is a loading control. (D) GCC185, GRP78, and ATF6 W-B of the lysates from the cells presented in A; β-actin is a loading control. (E) Calreticulin and HSP90 W-B of the lysates from the cells presented in A; β-actin is a loading control. (F and G) S1P (F) and S2P (G) W-B of the ER fraction isolated from the cells presented in A; HSP70 is a loading control. (H and I) IF staining of LNCaP cells to evaluate colocalization of S1P (red) and S2P (red) with GCC185 (green); bars, 10 μm. White boxes indicate area magnified at the right. (J) Quantification of the Pearson coefficient of colocalization for the cells presented in H and I (N = 90 cells from three repeats; **P < 0.001, t test). (K) AR W-B of the nuclear fraction from LNCaP cells: control, treated with ETOH, and ATF6 siRNA followed by EtOH. (L) HDAC6-P and HSP90-Ac W-B of the lysate of cells from K. (M) Anchorage-independent colony formation was measured by the soft agar assay for LNCaP cells: control, treated with ETOH, and ATF6 siRNA followed by EtOH; bars, 200 μm. (N) Quantification of the number of colonies in 20 randomly selected areas for cells from M (N = 3; **P < 0.001, t test). (O) Representative LNCaP xenograft tumors from control and EtOH-fed mice. (P) The tumor growth is presented as a ratio of tumor weight (g)/number of days after injection; medians (min – max); N = 9 mice from each group; ***P < 0.05, unpaired t-test). (Q) ATF6 immunostaining (red) of the tissue sections from xenograft tumors presented in O. (R) Quantification of the ratio of nuclear/cytoplasmic ATF6 IF in cells presented in Q (means ± SD; N = 3; **P < 0.001, t test). All images were acquired with the same imaging parameters