Journal: Toxicology

Article Title: Sertraline induces endoplasmic reticulum stress in hepatic cells

doi: 10.1016/j.tox.2014.05.007

Figure Lengend Snippet: Effects of brefeldin A and thapsigargin on the secretion of Gluc and SEAP proteins in HepG2 cells. As ER stress inducers, brefeldin A and thapsigargin were tested for their inhibitory effects on the molecules involved in ER stress in HepG2 cells and Gluc and SEAP secretion in the Gluc and SEAP reporter cells. (A and B) HepG2 cells were treated with various concentrations of brefeldin A and thapsigargin for 6, 16, and 24 h. The main ER stress response related proteins (PERK, CHOP, ATF4, and PDI) were determined by Western blotting. (C and D) After treatment with increasing concentrations of brefeldin A and thapsigargin for 24 h, the activities of Gluc and SEAP were determined as described in Section 2; the results shown are mean ± SD of four separate experiments. ###P < 0.001 represents relative SEAP activity was significantly different from the control; ***P <0.001 represents relative Gluc activity was significantly different form the control. Relative Gluc or SEAP activity was calculated based on % of each (Gluc or SEAP activity in medium per Fluc activity) over its vehicle control.

Article Snippet: Luciferase activity Gluc activity was measured by adding 50 μl Gaussia luciferase assay reagent (Nanolight Technology, Pinetop, AZ) to 5 μl of the conditioned cell-free medium and immediately determined the bioluminescence using a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT).

Techniques: Western Blot, Activity Assay, Control

Journal: Toxicology

Article Title: Sertraline induces endoplasmic reticulum stress in hepatic cells

doi: 10.1016/j.tox.2014.05.007

Figure Lengend Snippet: Effects of sertraline on the secretion of Gluc and SEAP proteins in HepG2 cells. (A) (HepG2-Gluc-Fluc) or (B) (HepG2-SEAP-Fluc) cells were treated with various concentrations of sertraline for 2 and 6 h. The activities of Gluc (A) or SEAP (B) was measured *P <0.05, **P <0.01, and ***P < 0.001 as compared with DMSO controls.

Article Snippet: Luciferase activity Gluc activity was measured by adding 50 μl Gaussia luciferase assay reagent (Nanolight Technology, Pinetop, AZ) to 5 μl of the conditioned cell-free medium and immediately determined the bioluminescence using a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT).

Techniques:

Journal: Toxicology

Article Title: Sertraline induces endoplasmic reticulum stress in hepatic cells

doi: 10.1016/j.tox.2014.05.007

Figure Lengend Snippet: Effect of silencing MAP4K4 on sertraline-induced ER stress. (A) Two HepG2 cell lines with silenced gene expression of MAP4K4 (sh1-MAP4K4 and sh2-MAP4K4) or a scramble control (SC) were treated with sertraline at 25 µM for 6 h. Total cellular proteins were extracted. The expression levels of MAP4K4 and ER stress related proteins (PERK, p-PERK, CHOP, PDI, and pro-caspase-4) were detected by Western blotting. GAPDH was used as a loading control B) After treatment with 10 µM SP600125 (JNK inhibitor) for 2 h (HepG2-Fluc-Gluc) cells were treated with indicated concentrations of sertraline for 6 h. The relative Gluc activity was calculated; the results shown are mean ± SD of four separate experiments. *P <0.05, **P <0.01 versus treatment with sertraline alone.

Article Snippet: Luciferase activity Gluc activity was measured by adding 50 μl Gaussia luciferase assay reagent (Nanolight Technology, Pinetop, AZ) to 5 μl of the conditioned cell-free medium and immediately determined the bioluminescence using a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT).

Techniques: Gene Expression, Control, Expressing, Western Blot, Activity Assay