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. 2020 Aug 14;18:126. doi: 10.1186/s12964-020-00609-7

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© The Author(s) 2020

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. 4 — Zyflamend-Induced Cell Death is Mediated through the ER Stress/Autophagy Pathways. Total cell lysates from control cells treated with Zyflamend and non-treated cells with or without AMPK inhibitor (Compound C: CC), (a-b), autophagy inhibitor (3-MA) (c-d), or ER stress inhibitor (4-PBA; e-h) were immunoblotted for markers of inflammation, ER stress, autophagy, and cell death. Cells were pre-treated with the indicated inhibitors 12 h prior to adding Zyflamend or DMSO (as a vehicle control) for an additional 24 h. In b, d, and f Bar graphs represent) Bar graphs represent pAMPK/AMPK, pPERK/PERK, pEIF2α/EIF2α, pIRE1α /IRE1α, sXBP1/β-actin, CHOP/β-actin, pJNK/JNK, Beclin 1/β-actin, LC3II&II/β-actin, and cleaved caspase3/β-actin as means + SEM. *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. †p < 0.05, ††p < 0.01 indicate a significant difference between cells treated with the indicated inhibitor and non-treated cells (CC, 3-MA, or 4-PBA). g Cells were treated with 200 μg/ml of Zyflamend for the indicated time with or without 4-PBA. Bar graphs represent the intensity of SRB staining reflective of the cell number and presented as means + SEM from at least three independent experiments. *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. †p < 0.05, ††p < 0.01 indicate a significant difference between non-treated and cells treated with 4-PBA. h-i Colony formation assay. i Bar graphs represent the relative number of colonies in each condition determined by dividing the number of colonies for a given treatment by the total number of colonies in DMSO treated cells (Ctrl.) and expressed as a percentage. j-k Chromatin Condensation in cells treated with 200 μg/ml of Zyflamend alone or in combination with 4-PBA for 24 h. Representative images are shown. Scale bar: 50 μm. i Bar graphs represent the number of apoptotic cells (Hoechst positive) as means + SEM from at least three independent experiments. In i and k, **p < 0.01 indicates a significant difference between cells treated with Zyflamend and non-treated cells. ††p < 0.01 indicates a significant difference between cells treated with 4-PBA and non-treated cells