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