Table 1.
Clinical information of patients involved in this study.
Table 2.
Comparison of mRNA and protein levels of the four genes with mitochondrial location.
Fig 1.
EFV inhibited cell proliferation and induced ROS production.
A) Huh-7 cells were incubated with EFV (2.5 and 10 mg/L) for 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 6.0, and 12.0 h, and detected by CCK8 kit. B) Huh-7 cells were incubated with the indicated concentrations of EFV for the indicated times. Production levels of ROS were detected with DCFH-DA probe. The cells were treated with EFV, NVP, TDF, H2O2 or rotenone for 2 h and analyzed the expression levels of ROS by flow cytometry. C) Total ROS was detected by DCFH-DA in Huh-7 cells. D) Mitochondrial ROS was detected by mitoSOX in Huh-7 cells. E) Total ROS in primary hepatocytes. F) Mitochondrial ROS levels in primary hepatocytes are shown. Data are the average of three independent experiments (total n = 3). Data shown are the mean ± SEM. ns, no significant difference, * p < 0.05 ** p < 0.01, and ***p<0.001.
Fig 2.
EFV induces alteration of the expression of 15 proteins in Huh-7 cells.
Huh-7 cells were treated with 2.5 and 10 mg/L EFV for 2 h. A) Enrichment of mitochondrial proteins is shown. The blots were probed with antibodies against organelle-specific proteins: anti-prohibitin for mitochondria, anti-Na+/K+-ATPase for plasma membrane; Homo, up and down mean homogenization, upper phase, and lower phase differences from control, EFV2.5 and EFV 10. B) Prohibitin-enriched upper phase was collected as mitochondria and analyzed by 2DE separation. The up-regulated proteins are shown. There were none down-regulated. C) Differentially expressed proteins were analyzed by Imagemaster software and shown as a bar graph. Data are representative of, or the average of, analyses of three independent experiments. The columns a, b & c represent control, d, e & f represent EFV 2.5, and g, h & i represent EFV 10.
Table 3.
List of protein spots identified by 2DE as differentially expressed after EFV treatment.
Fig 3.
MMAB levels were up-regulated in mitochondria by treatment with EFV.
Huh-7 cells were treated with 2.5 or 10 mg/L EFV for 2 h. A) Expression levels of GPD2, MMAB, NDUFS8 and SYNE2 mRNA. B) Expression levels of MMAB in mitochondria. Data are representative of, or the average of analyses of three independent experiments.
Fig 4.
Over-expression of MMAB in the liver of EFV-treated HIV-infected patients.
The liver sections from EFV-treated (EFV), or non-treated or PI-treated HIV patients (None) were stained with anti-MMAB monoclonal antibodies. A) Immunohistochemistry analysis. a, from patient without EFV treatment, b, from EFV treated patients. B) Quantitative analysis of MMAB expression in EFV (n = 5*10) or none (n = 6*10). Data are representative of, or the average of analyses of five samples from EFV-treated patients and 6 from patients without treatment or treated with PI drugs. Each sample was randomly scanned for 10 images, and used for statistic analysis.
Fig 5.
ROS production in Huh-7 cells after EFV treatment with or without MMAB knockdown.
The Huh-7 cells were transfected with MMAB-shRNA for 48 h and treated with EFV for additional 2 h. The ROS levels in the cells were measured by flow cytometry. Rotenone was used as positive control. *, **, *** or ****, and n.s. represent p < 0.05, 0.01, 0.001 and no significant difference, respectively.
Fig 6.
Upregulation of MMAB protein expression levels in Huh7 cells and primary hepatocyte cells by EFV treatment.
A) and C) Huh-7 cells; B) and D) primary hepatocyte cells (PHC). A) and B) were treated with 0.1% methanol (Control), EFV and H2O2 in Huh-7 and PHC for 2 h. C) and D) were treated with NVP, TDF, H2O2 for 2 h. EFV2.5 and EFV10 represent EFV treated at the concentration of 2.5 mg/L and 10 mg/L. *, **, *** or ****, and ns represent p < 0.05, 0.01, 0.001 and no significant difference, respectively.
Fig 7.
Enhancement of β-Ala production in Huh-7 cells with Knock-down of MMAB.
A) Huh-7 cells were transfected with MMAB-shRNA, and cultured for 48 h. B) and C) MMAB-siRNA transfected Huh-7 cells were analyzed for amino acid content and part of the amino acid profile is shown. The differently expressed amino acid (β-Alanine (b-Ala)) is highlighted by the arrow. B) is from control and C) is from shMMAB. D) and E) beta-Alanine metabolism pathway (from map00410 in KEGG) and protein interacting network with MMAB. The direct interacting protein ALDH3A2 is highlighted by “*” (D) and E)).
Fig 8.
Decreases of ALDH3A2 expression in Huh-7 cells with MMAB Knock-down.
Huh-7 cells were transfected with MMAB-shRNA, and cultured for 48 h. A) and B) The protein expressions of MMAB and ALDH3A2 detected by WB. B) statistical analysis in three replicate experiments. Optical density was analyzed by ImageJ software. Each band was read twice. Tubulin was used as reference protein. C) and D) The mRNA expressions of MMAB and ALDH3A2 detected by real-time RT-PCR. C) the fusion curve of MMAB, ALDH3A2 and 18S. D) statistical analysis in three replications. In each experiment, two parallel samples were loaded. 18S was used as reference gene.
Fig 9.
Proposed model for EFV-induced mitotoxicity based on previous studies and our results.