1. Volume 19, Issue 3, Pages 539-547 (March 2014)
High-Mobility Group Box 1 Is Dispensable for Autophagy, Mitochondrial Quality Control, and Organ Function In Vivo 
Peter Huebener, Geum-Youn Gwak, Jean-Philippe Pradere, Catarina M. Quinzii, Richard Friedman, Chyuan-Sheng Lin, Chad M. Trent, Ingmar Mederacke, Enpeng Zhao, Dianne H. Dapito, Yuxi Lin, Ira J. Goldberg, Mark J. Czaja, Robert F. Schwabe 
Cell Metabolism 
Volume 19, Issue 3, Pages (March 2014)
DOI: /j.cmet
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2. Cell Metabolism 2014 19, 539-547DOI: (10.1016/j.cmet.2014.01.014)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Mice with Hepatocyte-Specific Deletion of Hmgb1 Develop Normally and Do Not Display Significant Alterations in Hepatic Gene Expression
(A) Growth curves of Hmgb1f/f and Hmgb1Δhep mice do not diverge (n ≥ 6 mice per group and time point).
(B) Quantification of Hmgb1 deletion via qPCR (n = 4 per group) and western blot analysis in whole-liver extracts reveals efficient reduction of Hmgb1 levels in Hmgb1Δhep livers (−90% and −72%, respectively, both p < 0.001).
(C) Immunohistochemical staining demonstrates HMGB1 deletion in hepatocytes, but not in nonparenchymal cells (arrows) in Hmgb1Δhep livers.
(D) Microarray heatmap of Hmgb1f/f and Hmgb1Δhep livers, showing significant changes of Hmgb1 and only six additional probesets in a total of >30,000 probesets in Hmgb1Δhep livers.
(E) qPCR shows similar hepatic mRNA levels of albumin, AFP, and inflammatory and proliferative genes in Hmgb1f/f and Hmgb1Δhep mice (n = 3/group).
(F and G) No differences in liver injury and steatosis in 8-week-old mice (n = 5/group) were shown, according to serum ALT levels (F) and hepatic triglyceride content (G).
(H) Hematoxylin and eosin (H&E)-, oil red O-, periodic acid-Schiff (PAS)-, and TUNEL-stained sections of livers from 8-week-old mice reveal no differences in liver architecture, fat and glycogen content, or apoptosis in the absence of hepatocyte HMGB1.
Scale bars, 20 μm (C) and 50 μm (H). Data are represented as mean ± SD. ∗∗p < 0.01; n.s., nonsignificant. See also Figure S1 and Table S1.
Cell Metabolism  , DOI: ( /j.cmet )
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4. Figure 2
Liver-Specific Hmgb1 Deletion Does Not Affect Mitochondrial Function
(A and B) Blood glucose before and after 24 hr of starvation (A) and in response to glucagon administration (16 ng/g i.p. following 6 hr of starvation) (B) demonstrate normal regulation of glucose homeostasis in the absence of hepatocyte HMGB1.
(C and D) No differences are revealed between Hmgb1f/f (n = 5) and Hmgb1Δhep (n = 5) mice according to enzymatic activity analysis of key respiratory chain enzymes (C), and histochemical activity stainings for respiratory chain complexes II (COX) and IV (SDH) (D).
(E and F) Quantification of whole-liver ATP levels (E) and ATP/ADP ratio (F) as end points of energy substrate generation demonstrate completely preserved mitochondrial function and ATP generation in Hmgb1Δhep livers (n = 5/group).
(G) Whole-body metabolic assessment of 4-month-old male Hmgb1f/f and Hmgb1Δhep mice (n = 4 per group) reveals similar activity levels, energy expenditure, and respiratory exchange rates in mice from both groups.
(H) Real-time assessment of oxidative phosphorylation using an extracellular flux analyzer after sequential addition of oligomycin (2 μM), FCCP (1 μM), 2-DG (100 mM), and rotenone (1 μM) demonstrates similar basal OXPHOS, glycolysis and reserve capacity, and a similar glutamine-induced increase of OCR in isolated primary hepatocytes from Hmgb1Δhep and Hmgb1f/f mice (n = 5 isolations each). Scale bars, 100 μm. Data are represented as mean ± SD n.s., nonsignificant.
Cell Metabolism  , DOI: ( /j.cmet )
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5. Figure 3
Mitochondrial Structure and Mitophagy Are Preserved in Mice with a Hepatocyte-Specific Hmgb1 Deletion
(A) Electron microscopy of liver sections shows normal mitochondrial ultrastructure in Hmgb1Δhep livers.
(B–D) Primary hepatocytes from Hmgb1f/f LC3-GFP-positive (n = 3) and Hmgb1Δhep LC3-GFP-positive double-transgenic mice (n = 3) loaded with MitoTracker Red (100 nM) both show regularly shaped mitochondria, with only few GFP-positive puncta under baseline conditions (B). Stimulation of the cells with glucagon (2 μM for 90 min) or rotenone (1 μM for 6 hr) results in a robust induction of GFP-positive organelles (lysosomes) (C) that partially colocalize with MitoTracker-stained mitochondria (inlays) (D).
(E and F) Starvation for 24 hr of Hmgb1f/f LC3-GFP-positive (n = 3) and Hmgb1Δhep (n = 3) LC3-GFP-positive double-transgenic mice results in similar patterns of LC3-GFP expression in the liver, as assessed by confocal microscopy (E) and similar increases in LC3-GFP cleavage (F).
Scale bars, 2 μm (A), 10 μm (B), and 50 μm (E). Data are represented as mean ± SD n.s., nonsignificant. See also Figure S2.
Cell Metabolism  , DOI: ( /j.cmet )
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6. Figure 4
Cardiomyocyte-Specific Deletion of Hmgb1 Does Not Affect Mitochondrial Structure and Organ Function
(A) Growth curves of Hmgb1f/f and Hmgb1CM mice do not diverge (n = 5 mice per group and time point).
(B and C) Efficient deletion of Hmgb1 from Hmgb1ΔCM hearts is demonstrated by qPCR and western blot (B) and immunohistochemistry (C) (n = 4/group).
(D) Electron microscopy demonstrates comparable mitochondrial morphology in Hmgb1f/f and Hmgb1ΔCM cardiomyocytes.
(E) Quantitative assessment of cardiac ATP levels, ATP/ADP ratios, and respiratory chain enzyme activities reveal normal mitochondrial function in the absence of cardiomyocyte HMGB1.
(F) Histochemical activity staining for respiratory chain complexes II (COX) and IV (SDH) activities reveals no differences between Hmgb1f/f and Hmgb1ΔCM mice.
(G) Echocardiographic assessment of cardiac architecture and function reveals normal ejection fraction, fractional shortening, heart rate, and relative wall thickness in 4-month-old Hmgb1ΔCM mice (n = 5/group).
(H and I) Similar Hspb1 expression in the heart, irrespective of the HMGB1 status, as demonstrated by qPCR (H) and western blot analysis (I) (n = 4/group).
Scale bars, 50 μm (C), 2 μm (D), and 100 μm (F). Data are represented as mean ± SD. ∗p < 0.05; ∗∗p < 0.01; n.s., nonsignificant. See also Figure S3.
Cell Metabolism  , DOI: ( /j.cmet )
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