Fig 1.
VPA and SAHA reinforce I/R-mediated hyperacetylation of Histone H3.
A) Time-schedule of the performed experiments. VPA: valproic acid; SAHA: suberoylanilide hydroxamic acid; DMSO: vehicle dimethylsulfoxide. B) Western blot for acetylated Lysine 9 of Histone H3. β-actin served as loading control. Representative blots for 6h and 24h of reperfusion are shown. Bar graphs depict quantification of at least four experiments. NI: non-ischemic right lobes; I: ischemia-reperfusion left lobes. ***: p<0.001; **: p<0.01.
Fig 2.
VPA- and SAHA-treatment delays I/R-damage but fails to exert protective properties.
A) Serum levels of aspartate (AST) and alanine (ALT) aminotransferases at different time points. B) Representative H/E micrographs of left lateral liver lobe sections from animals treated as indicated. On the images with lower magnification (x40, scale bars 200μm) damaged areas are encircled. The images with higher magnification (lower row of 6h reperfusion; x400, scale bars 20μm) depict sinusoidal jamming (star) and signs of hepatocyte cell death like karyopyknosis (thick arrow), karyorrhexis (arrowhead), karyolysis (fine arrow) and loss of nuclei (raindrop) for all treatment options as indicated. C) Histological quantification of tissue damage. One whole representative horizontal section per animal was analyzed. Percentage of damaged area (includes: sinusoidal jamming, cell death/scar and inflammatory infiltrates) is given. D) Representative photographs of whole liver (top row), and dissected left lateral and left part of median liver lobe (bottom row) of animals subjected to the indicated treatments and sacrificed at indicated timepoints. E) Quantification of I/R-damage by macroscopic aspect of the left lateral liver lobe. Integrated volume of necrosis/scar was set in relation to the calculated total volume of the lobe. *: p<0.05. TTP: time to peak; AUC: area under the curve.
Fig 3.
VPA and SAHA modulate I/R-dependent activation of p38- and JNK-signaling.
A) Representative Western blots for (phospho-)JNK (T183/T185) and (phospho-)p38 (T180/Y182). β-actin served as loading control. B) Densitometric blot-quantifications for indicated treatment groups and reperfusion periods of at least three experiments. NI: non-ischemic right lobes; I: post-ischemic I/R left lobes. **: p<0.01; *: p<0.05.
Fig 4.
Impact of HDAC-inhibition on regeneration.
A) Ki67 immunohistochemistry. Representative micrographs of non-damaged and damaged areas in sections of post-ischemic left liver lobes and of non-ischemic control lobes from animals treated as indicated. Scale bars: 200μm. B) Quantification of Ki67-positive hepatocytes in I/R unaffected areas. At least 20 HPFs of areas without massive inflammatory infiltrates and scar tissue were counted per slide. C) Representative Western blots with (phospho-)YAP (S127) antibodies and D) Graphs of densitometric quantifications of at least three experiments. Values were set in relation to the samples of sham treated animals. E) YAP immunohistochemistry. Representative micrographs of non-damaged and damaged areas in sections of post-ischemic left liver lobes and of non-ischemic right control lobes from animals treated as indicated. White arrows indicate nuclear staining in the images for non-ischemic lobes and the non-damaged areas of post-ischemic lobes. Arrows were omitted in images for damaged areas of post-ischemic lobes. Scale bars: 100μm. F) Quantification of nuclear YAP-positive hepatocytes. At least 20 HPFs were counted per slide. NI: non-ischemic right lobes; I: post-ischemic I/R left lobes. **: p<0.01; *: p<0.05. AUC: area under the curve.