Article Figures & Data
Figures
- Fig. 1.
Aripiprazole, but not Olanzapine, induces ER stress in HepG2 cells. (A) HepG2 cells were treated overnight with the indicated concentrations of Ari. Total cell extracts of the adhered cells were analyzed by immunoblotting for phosphorylated IRE1, total IRE1, total PERK, and p97 as loading control. The lower panel shows RT-PCR analysis of XBP1 splicing after Ari treatment. A representative experiment of three independent repetitions is shown. (B) Relative quantification of phosphorylated IRE1 (24 hours). Shown is an average of three independent experiments ± S.D. (C) Time course of IRE1 phosphorylation after Ari treatment. Tg (2.5 µg/ml) was used as a positive control. (D) Similar analysis as in (A) was conducted for Ola. Statistical significance is indicated by **P < 0.01; ***P < 0.001 by ANOVA.
- Fig. 2.
Aripiprazole promotes immobilization of calcium to the cytoplasm. (A) The timeline of the experimental procedure. Tg is used twice as a positive control and to release ER calcium storage immediately before imaging. (B) Pseudocolored images of calcium concentrations of HepG2 cells before (“basal”) and after Tg (2.5 µg/ml) application to release the ER calcium storages after overnight treatment with DMSO as a negative control, Tg (2.5 µg/ml) as a positive control, or Ari. Scale bar indicates levels of intracellular calcium. (C) Changes of intracellular calcium levels in HepG2 cells treated as shown in (A). Statistical significance is indicated by *P < 0.05 of at least five independent measurements by ANOVA.
- Fig. 3.
IRE1 contributes more than PERK to Ari-induced cytotoxicity. (A) WT, IRE1 KO, and PERK KO HepG2 were treated with DMSO and 30 µM of Ari. Cells were stained with PI and analyzed by flow cytometry. Representative dot plots of forward vs. side scattering properties and percentage of PI-positive cells are shown. (B) Percentage of PI-positive cells, measured by flow cytometry, after overnight treatment with DMSO, Ari (30 µM), and Ari with Z-VAD-FMK (25 µM). Average of three independent experiments ± S.D. is shown. Statistical significance is indicated by *P < 0.05; **P < 0.01 by ANOVA for Z-VAD-FMK treatment and for the KO cells by paired Student’s t test.
- Fig. 4.
N-acetyl cysteine (NAC), BAPTA-AM, and EGTA do not protect from Aripiprazole-induced cell death. (A) HepG2 cells were treated overnight with DMSO and 30 µM of Ari in the absence or presence of NAC, BAPTA-AM, or EGTA. Percentage of PI-positive cells of a representative experiment, as measured by flow cytometry after treatment, is shown. (B) Average of three independent experiments ± S.D. is shown. No statistical significance was measured.
- Fig. 5.
GSH ethyl ester protects from Aripiprazole-induced cell death. HepG2 cells were treated overnight with DMSO and 30 µM of Ari in the absence or presence of GSH ethyl ester. Percentage of PI-positive cells of a representative experiment, as measured by flow cytometry after treatment, is shown. Average of three independent experiments ± S.D. is shown. Statistical significance is indicated by *P < 0.05 by ANOVA.
- Fig. 6.
Aripiprazole induces ER stress, and accumulation of lipids and calcium mobilization form the ER in primary mouse hepatocytes. (A and B) Primary mouse hepatocytes were isolated and treated with Ari at different concentrations. Four (A) and 8 hours (C) after exposure to the drug, cells were lysed and analyzed by immunoblotting for phosphorylated IRE1, total IRE1, total PERK, and p97 as loading control. Four (B) and 8 hours (D) relative quantification of phosphorylated IRE1. Shown is an average of three independent experiments ± S.D. (E) RT-PCR analysis for XBP1 mRNA splicing after Ari treatment. Tm (2.5 µg/ml) was used as a positive control for ER stress induction. (F) Oil Red-O staining of primary mouse hepatocytes after 24 hours of treatment with DMSO, Tm (2.5 µg/ml), or 30 µM of Ari. Representative phase contrast images are shown. (G) Pseudocolored images of calcium concentrations of primary hepatocytes before (“Basal”) and after Tg (2,5 µg/ml) application to release the ER calcium storages after overnight treatment with DMSO as a negative control, Tg (2.5 µg/ml) as a positive control, or 30 µM of Ari. Scale bar indicates levels of intracellular calcium. (H) Changes of intracellular calcium levels in primary hepatocytes treated as shown in (G). Statistical significance is indicated by **P < 0.01 by ANOVA.
Additional Files
Data Supplement
- Supplemental Figures -
Figures S1-S8.
- Supplemental Figures -
