Fig. 3.

Zyflamend Induces Inflammatory, ER Stress, and Autophagy Responses in β-TC6 Cells. a-b Total cell lysates from control and Zyflamend treated cells for 3, 6, 12, 24, 36 and 48 h were immunoblotted for ER stress markers: pPERK, pEIF2α, pIRE1α, their respective unphosphorylated proteins, sXBP1, CHOP, and β-actin as a loading control. Representative immunoblots are shown. b Bar graphs represent pPERK/PERK, pEIF2α/EIF2α, pIRE1/IRE1, sXBP1/β-actin, and CHOP/β-actin as means + SEM. *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. c-d Markers of autophagy were examined in the same lysates using antibodies against Beclin 1, LC3, I&II, ATG5, ATG7, and β-actin as a loading control. d Bar graphs represent Beclin 1/β-actin, LC3/β-actin, ATG5/β-actin, and ATG7/β-actin as means + SEM. *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. e-f Immunoblots of key proteins in autophagy, AMPK, ER stress, and apoptosis signaling in β-TC6 cells treated with 200 μg/ml Zyflamend with and without the pan-caspase inhibitor Z-VAD.fmk. Representative immunoblots are shown. f Bar graphs represent pAMPK/AMPK, pPERK/PERK, pEIF2α/EIF2α, pIRE1α/IRE1α, sXBP1/β-actin, CHOP/β-actin, pJNK/JNK, Beclin 1/β-actin, LC3I&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 Z-VAD.fmk and non-treated cells