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Additional Supporting Information may be found in the online version of this article.

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HEP_24690_sm_SuppFig1.tif213KSupplemental Figure 1. No obvious necrotic cell death is induced by various concentrations of APAP after 6 hours treatment in primary mouse hepatocytes. (A) Primary cultured mouse hepatocytes were treated with various concentrations of APAP (0, 1.25, 2.5, 5 and 10 mM) for 6 hrs followed by PI staining. PI positive cells with intact nuclei were quantified for each experiment (n=3). More than 300 cells were counted in each individual experiment. (B) Primary mouse hepatocytes were treated with APAP (5 Mm) for 6 hrs and then processed for EM analysis. Representative EM images of autophagosomes containing mitochondria in APAP-treated hepatocytes were shown.
HEP_24690_sm_SuppFig2.tif242KSupplemental Figure 2. Suppression of autophagy enhances APAP-induced ROS production. Primary cultured mouse hepatocytes were treated with APAP (5 mM) in the absence or presence of CQ (20 μM) for 6 hrs. The cells were stained with DCFH-DA (2.5 μM) for 15 min and washed two times with PBS followed by fluorescence microscopy. Representative photographs were shown in (A). (B) Quantification of DCF fluorescence intensity. Data are means ± SD from three independent experiments.
HEP_24690_sm_SuppFig3AandC.tif3793KSupplemental Figure 3. APAP-induced necrosis in hepatocytes is suppressed by rapamycin. (A) Representative overlayed images of phase-contrast with PI staining of primary mouse hepatocytes treated for 24 hrs. Panel a: non-treated; panel b: TNF-α (10 ng/mL) plus ActD (0.1 μg/mL); panel c: APAP (10 mM). Panel d: enlarged photograph from the boxed area in panel c. (B) PI positive cells were quantified for each experiment (n=3). More than 300 cells were counted in each individual experiment *: p<0.01. (C) Representative EM images of control and APAP-treated hepatocytes for 24 hrs were shown. N: nucleus; m: mitochondria. (D) Primary mouse hepatocytes were co-treated for 24 hrs as indicated: panel a: DMSO; panel b: APAP (10 mM)+ DMSO (4 μL/mL); panel c: APAP (10 mM) + Rap (2 μM); and panel d: Rap (2 μM). Representative overlayed images of phase-contrast with PI staining were shown.
HEP_24690_sm_SuppFig3D.tif2136KSupplemental Figure 3. APAP-induced necrosis in hepatocytes is suppressed by rapamycin. (A) Representative overlayed images of phase-contrast with PI staining of primary mouse hepatocytes treated for 24 hrs. Panel a: non-treated; panel b: TNF-α (10 ng/mL) plus ActD (0.1 μg/mL); panel c: APAP (10 mM). Panel d: enlarged photograph from the boxed area in panel c. (B) PI positive cells were quantified for each experiment (n=3). More than 300 cells were counted in each individual experiment *: p<0.01. (C) Representative EM images of control and APAP-treated hepatocytes for 24 hrs were shown. N: nucleus; m: mitochondria. (D) Primary mouse hepatocytes were co-treated for 24 hrs as indicated: panel a: DMSO; panel b: APAP (10 mM)+ DMSO (4 μL/mL); panel c: APAP (10 mM) + Rap (2 μM); and panel d: Rap (2 μM). Representative overlayed images of phase-contrast with PI staining were shown.
HEP_24690_sm_SuppFig4.tif211KSupplemental Figure 4. Rapamycin does not affect the initial glutathione (GSH) depletion induced by APAP. (A) Wild type C57BL/6 mice were injected (i.p.) with rapamycin (2 mg/kg) or DMSO (2% DMSO, 10 μL/g) immediately followed by APAP (500 mg/kg) injection for 2 hrs. Blood ALT levels were quantified (n=3 mice). (B) The liver content of total GSH was determined as described previously4 (C) Wild type C57BL/6 mice were injected (i.p.) with CQ (60 mg/kg), rapamycin (2 mg/kg) or DMSO (2% DMSO, 10 μL/g) immediately followed by APAP (500 mg/kg) injection for 6 hrs. The liver content of total GSH was then determined.
HEP_24690_sm_SuppInfo1.doc45KSupporting Information without line.
HEP_24690_sm_SuppInfo2.doc44KSupporting Information

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