1. Volume 4, Issue 3, Pages 185-198 (September 2006)
Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis 
Montserrat Marí, Francisco Caballero, Anna Colell, Albert Morales, Juan Caballeria, Anna Fernandez, Carlos Enrich, José C. Fernandez- Checa, Carmen García-Ruiz 
Cell Metabolism 
Volume 4, Issue 3, Pages (September 2006)
DOI: /j.cmet
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Diet-induced hepatic steatosis
A) Representative hematoxylin and eosin (H&E) (magnification at 10×) or Oil Red O-stained 4-micron sections (magnification at 20×) of liver sections from chow-, Lombardi-, or HC-fed rats for 2 days. L-HC animals were fed Lombardi diet for 3 days plus HC diet for 2 days.
B–D) Hepatic cholesterol, TG, and FFA concentrations were measured in liver homogenate from animals fed the different diets for 2 days. All values are expressed as mean (± SEM), n = 8–12. ∗p < 0.05 versus chow-fed group.
E) Representative fluorescence microscopy of free cholesterol by filipin (0.05 mg/ml) staining of cultured hepatocytes isolated from all groups as described in Experimental Procedures.
Cell Metabolism 2006 4, DOI: ( /j.cmet )
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3. Figure 2 Sensitization of steatotic hepatocytes to TNF
A) Cell death was determined 16 hr after TNF (100 ng/ml) challenge by double staining with Höechst (10 μM) and propidium iodide (10 μM) to detect apoptotic and necrotic cells, respectively. At least 250 cells in six different high-power fields were counted and expressed as a percentage of total cells (mean ± SD).
B) Cytochrome c release was determined by Western blot 6 hr after TNF challenge in cytosol (spnt) and mitochondria (pellet). β-actin was used as a loading control.
C) 6 hr after TNF treatment, aliquots of cell extracts were prepared for caspase 3 activity using a fluorescent peptide. ∗p < versus chow-fed hepatocytes. The data represent the mean ± SEM.
D) ROS were determined 4 hr after TNF challenge in hepatocytes by DCF fluorescence. ∗p < 0.01 versus chow-fed hepatocytes. The data represent the mean ± SEM.
E and F) Serum ALT and AST were determined 24 hr after intravenous LPS injection. Mean ± SEM values are shown, n = 8–12 animals per group. ∗p < versus chow-fed group.
G) Representative H&E slides from rats after the LPS challenge or mice 8 hr after an i.p. injection of Jo2 (5 μg/mouse). H&E-stained sections were photographed on a Zeiss Axioplan using a Nikon DXM1200F digital camera (magnification at 20×). The serum ALT values (IU/L) correspond to the Jo2 treatment, ∗p < 0.05 versus chow-fed mice.
H) Representative myeloperoxidase staining of liver sections in HC-fed livers challenged with LPS (magnification at 40×).
Cell Metabolism 2006 4, DOI: ( /j.cmet )
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4. Figure 3 Mitochondrial FC accumulation depletes mGSH
A) Western blot of cytochrome c, GRP78/Bip, Na+/K+ATPase α1, Rab5A, and Rab11 in liver homogenates or mitochondrial fraction.
B) Purified mitochondria were fixed and processed for electron microscopy (17,500×).
C and D) Purified rat liver mitochondria were analyzed for the total as well as free and esterified cholesterol content. (C) Total cholesterol from chow (n = 8), HC (n = 12), and Lombardi (n = 8) animal feeding are shown. The data represent the mean ± SD. (D) Free and esterified cholesterol levels in mitochondria from HC-fed rats were analyzed by HPLC and mean ± SD values are shown.
E) Hepatocytes from chow- or HC-fed rats for 1 day were isolated to examine the colocalization of mitochondria and FC by confocal microscopy using mouse anti-cytochrome c Ab and filipin, respectively.
F) Purified rat liver mitochondria were labeled with DPH or TMA-DPH and fluorescence anisotropy was monitored at 366 nm (emission = 440 nm) using polarizing filters in both excitation and emission planes and normalized per mg of mitochondrial protein. Mean ± SD values from 4 rats/group are shown. ∗p < 0.01 versus chow-fed group.
G) Compartmentalized GSH from freshly isolated hepatocytes from the various groups were analyzed by HPLC as described in Experimental Procedures. Values are expressed as mean ± SD, n = 12 rats/group. ∗p < versus control hepatocytes.
Cell Metabolism 2006 4, DOI: ( /j.cmet )
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5. Figure 4
Hepatocytes from NPC1−/− mice exhibit mitochondrial FC loading and mGSH depletion
A) Hepatic cholesterol, TG, and FFA concentrations were measured in liver homogenates. All values are expressed as mean (± SEM), n = 5. ∗p < 0.05 versus chow-fed group.
B and C) Mouse hepatocytes from wild-type or NPC1−/− mice were isolated and stained with filipin, mouse anti-cytochrome c, or rabbit anti-Bip/GRP78 followed by the appropriate secondary antibodies. Experiments shown are representative confocal images of three to four different experiments.
D) Cell death was determined by double staining with Höechst (10 μM) and propidium iodide (10 μM) to detect apoptotic and necrotic cells, respectively. Values are expressed as mean ± SD of at least three experiments. ∗p < 0.01 versus control WT hepatocytes.
E) compartmentalized GSH from freshly isolated WT and NPC1−/− hepatocytes were analyzed by HPLC as described in Experimental Procedures. Results are mean ± SD of at least three experiments. ∗p < 0.01 versus WT hepatocytes.
Cell Metabolism 2006 4, DOI: ( /j.cmet )
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6. Figure 5 mGSH depletion in vitro by HP sensitizes hepatocytes to TNF
A) GSH compartmentalization in cytosol and mitochondria from hepatocytes treated with HP (0.5 mM) for 5 min. Results are expressed as mean ± SD of at least six experiments. ∗p < 0.01 versus untreated hepatocytes.
B) Control or HP-treated mouse hepatocytes were exposed to TNF for 16 hr with or without caspase 8 inhibitor, BHT, or vitamin E, as described in Experimental Procedures. Cell death was determined as in Figure 2A. At least 250 cells in six different high-power fields were counted and expressed as a percentage of total cells (mean ± SD). ∗p < 0.01 versus control.
C) ROS were monitored over time after TNF challenge in hepatocytes. Results are mean ± SD of four experiments. ∗p < 0.01 versus control.
D) ROS were determined 4 hr after TNF challenge in hepatocytes isolated from wild-type and ASMase−/− mice. The data represent the mean ± SD.
E) ASMase−/− mouse hepatocytes were exposed to TNF for 16 hr after mGSH depletion by HP. Cell death was determined as in panel (B). The data represent the mean ± SD.
F) Representative NF-κB mobility shift assay using nuclear extracts from hepatocytes after TNF exposure.
G) Representative RT-PCR of iNOS and cIAP1, as described in Experimental Procedures.
Cell Metabolism 2006 4, DOI: ( /j.cmet )
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7. Figure 6 SAM prevents LPS-induced NASH
A) Representative H&E staining of liver samples from HC-fed rats 24 hr after LPS injection plus or minus SAM treatment.
B) Serum ALT and AST 24 hr after intravenous LPS injection plus or minus SAM in HC-fed animals. The data represent the mean ± SEM.
C) GSH levels in mitochondria isolated from chow- or HC-fed rats for 1 day and then treated with LPS plus or minus SAM for 24 hr. Results are expressed as Mean ± SEM, n = 8–12 animals per group. ∗p < 0.01 versus chow group. ∗∗p < 0.05 versus HC+LPS group.
Cell Metabolism 2006 4, DOI: ( /j.cmet )
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8. Figure 7 Cholesterol in ob/ob mice and atorvastatin therapy
A and B) Mouse hepatocytes from wild-type or ob/ob mice were isolated and stained with filipin, mouse anti-cytochrome c, or (B) rabbit anti-Bip/GRP78 followed by the appropriate secondary antibodies. Experiments shown are representative confocal images of three to four different experiments performed.
C) GSH in cytosol and mitochondria from chow or ob/ob hepatocytes. Results are expressed as mean ± SD of at least three experiments.
D) Serum ALT and AST levels in ob/ob or lean mice following or not atorvastatin (Atorv) therapy and/or LPS challenge. Data are mean ± SEM. n = 6 mice per group. ∗p < 0.05 versus untreated ob/ob mice; #p < 0.05 versus atorvastatin-treated ob/ob mice; ∗∗p < 0.05 versus LPS-treated ob/ob mice.
E) Representative H&E images from ob/ob mice after atorvastatin therapy or not and/or LPS challenge (magnification at 20×).
Cell Metabolism 2006 4, DOI: ( /j.cmet )
Copyright © 2006 Elsevier Inc. Terms and Conditions