Initially, in vitro experiments have been performed with HepG2 cells to establish the

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Valentine Burke
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1 1 SUPPLEMENTARY MATERIAL AND METHODS Cells and cell culture Initially, in vitro experiments have been performed with HepG2 cells to establish the in vitro model. This cell line has been most extensively employed in many studies analyzing drug metabolism and toxicity, since the cells persist a large part of cellular functions similar to those of normal hepatocytes [1,2]. Also because of the high degree of morphological and functional differentiation, HepG2 cells are a suitable model to study intracellular trafficking and drug targeting in vitro [3]. Subsequently, experiments have been repeated with primary human hepatocyts (PHH) confirming the data obtained with HepG2 cells. Analysis of cellular lipid content Cellular lipid droplets were visualized by Oil Red O staining as described [4]. Total cellular triglycerides and free fatty acids (FFA) were extracted using the method of Bligh and Dyer with slight modifications [5] and quantified by the triglyceride determination kit (GPO) (Sigma, Deisenhofen, Germany) as described [5] and free fatty acids-half micro test kit (Roche, Penzberg, Germany) following the manufacturer s instructions, respectively. Quantification of oil red O staining Quantification of staining intensity of lipid droplets was performed using a light microscope with digitalized camera and a Metaview image analysis system. The density of lipid droplets per microscopic area was calculated from four different fields.

2 2 Analysis of lipid peroxidation One of the major secondary products of lipid peroxidation is malondialdehyde (MDA), which forms a 1:2 adduct with thiobarbituric acid. Cellular MDA levels were analyzed using the OxiSelect-thiobarbituric acid-reactive substance (TBARS) assay kit (Cell Biolabs, San Diego, CA, USA) following the manufacturer s instructions. Quantitative Real-Time-PCR Analysis RNA isolation from cultured cells and reverse transcription were performed as described [6]. Quantitative real-time-pcr was performed applying LightCycler technology (Roche) [6] and the following pairs of primers: human ICAM-1 (forward: 5 -CTGTCACTCGAGATCTTGAGG-3 ; reverse: 5 -CCTGCAGTGCCCATTATGA-3 ), human IL-8 (forward: 5 -TCTGCAGCTCTGTGTGAAGGTGCAGTT-3 ; reverse: 5 - AACCCTCTGCACCCAGTTTTCCT-3 ), human IL-1ß (forward: 5 GCA CGA TGC ACC TGT ACG AT-3 ; reverse: 5 -CAC CAA GCT TTT TTG CTG TGA GT-3 ), human CCL2 (forward: 5 -CGC GAG CTA TAG AAG AAT CAC-3 ; reverse: 5 -TTG GGT TGT GGA GTG AGT GT-3 ) and murine CCL2 (forward: 5 TGC AGG TCC CTG TCA TGC TTC-3 ; reverse: 5 -TGG ACC CAT TCC TTC TTG GGG T-3 ). All other mrna expression analyses were performed using QuantiTect Primer Assays according to the manufacturer s instructions (Qiagen, Hilden, Germany). Amplification of cdna derived from 18s rrna (forward: 5 -AAACGGCTACCACATCCAAG-3 ; reverse: 5 - CCTCCAATGGATCCTCGTTA-3 ) was used for normalization. Protein analysis Protein extraction and Western blotting was performed as described [7] applying antirabbit antibodies against LC3I/II from Novus Biologicals (NB ), phosphop44/42 MAPK(ERK1/2) (#9101), p62 (#5114) and phospho-p38 (#9215) all from Cell

3 3 Signaling Technology (Danvers, MA, USA; all diluted 1:1,000). For densitometric analysis, Licor Image Studio Lite software (Licor) was used. (Immuno) histological analyses For histological analysis murine liver tissue specimens were fixed for 24 h in 4% formalin at room temperature, dehydrated by graded ethanol and embedded in paraffin. Tissue sections (thickness 5 µm) were deparaffinized with xylene and stained with HE or Sudan red for lipid droplets detection. Immunohistochemical staining was performed using anti-cd3 C7930 antibody (1:500; Sigma-Aldrich) as described [8]. LC3 Immunofluorescence Was performed as described in [9] with slight modifications. Briefly, HepG2 cells were stimulated for 12 h with IX (25 and 50 µm). Subsequently, cells were washed with PBS, and then fixed with 4% Paraformaldehyde for 10 min. After blocking for 30 min with 5% bovine serum albumin (BSA), cells were incubated with rabbit anti-lc3 antibody (NB ) diluted 1:200 in 5% BSA. Slides were washed three times with PBS before 60-min incubation with alexa fluor 546-conjugated goat anti-rabbit IgG antibody (diluted 1:200 in 5% BSA) (ThermoFisher). The LC3 puncta were visualized with a fluorescence light microscope. Ratiometric ph measurements with LysoSensor Yellow/ Blue DND-160 HepG2 cells were seeded 18 h before imaging at 2x10 5 in 35 mm glass bottom culture dishes (MatTek). The cells were incubated (or treated) for 30 min-2 h with irinotecan (IX) together with 1 µm LysoTracker Red DND-99 (life technologies) in culture medium to stain acidic compartments (e.g. lysosomes). After washing with

4 4 equilibration buffer (5 mm NaCl, 115 mm KCl, 1.2 mm MgSO4, 25 mm MES; ph 6.0) the cells were incubated in 2 μm Lysosensor Yellow/Blue DND-160 (Life Technologies) in equilibration buffer and life cell images were acquired between 1-15 min after addition of the dye with an ApoTome (Zeiss) and AxioVision 4.8 software. Cells were excited with 365 nm and the emission filters were 445 nm (B, less acidic) and 510 nm (Y; more acidic). Mean pixel values of fluorescence intensities of regions of interest (ROIs, 10 μm 2 ) corresponding to lysosomes (red fluorescence) were measured using AxioVision 4.8 software. Yellow to blue ratio of fluorescence intensities was further used to calculate vesicular ph employing the potential fit parameters from the calibration curve. To calibrate Lysosensor Yellow/Blue DND-160 emission ratios for a range of ph values in situ, cells were incubated before addition of the dual-wavelength fluorophore for 15 min with equilibration buffer of known ph ( ) in the presence of 10 μm monensin, a Na + /H + antiporter, and 10 μm nigericin, a K + /H + antiporter, to bring the cytosol and chromaffin vesicles to the ph of the surrounding media. A standard curve was generated in Excel to assign a predicted ph for relevant Y/B values. An independent standard curve was generated on each experimental day. Yellow/blue values from duplicate groups of 20 individual cells in two independent experiments were averaged for the analysis.

5 5 REFERENCES 1 Dehn PF, White CM, Conners DE, et al. Characterization of the human hepatocellular carcinoma (hepg2) cell line as an in vitro model for cadmium toxicity studies. In vitro cellular & developmental biology Animal 2004;40: Roe AL, Snawder JE, Benson RW, et al. HepG2 cells: an in vitro model for P450- dependent metabolism of acetaminophen. Biochemical and biophysical research communications 1993;190: Xia JF, Gao JJ, Inagaki Y, et al. Flavonoids as potential anti-hepatocellular carcinoma agents: recent approaches using HepG2 cell line. Drug Discov Ther 2013;7: Wobser H, Dorn C, Weiss TS, et al. Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells. Cell research 2009;19: Buettner R, Newgard CB, Rhodes CJ, et al. Correction of diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance by moderate hyperleptinemia. American journal of physiology Endocrinology and metabolism 2000;278:E Hellerbrand C, Muhlbauer M, Wallner S, et al. Promoter-hypermethylation is causing functional relevant downregulation of methylthioadenosine phosphorylase (MTAP) expression in hepatocellular carcinoma. Carcinogenesis 2006;27: Gabele E, Dostert K, Dorn C, et al. A new model of interactive effects of alcohol and high-fat diet on hepatic fibrosis. Alcoholism, clinical and experimental research 2011;35: Dorn C, Engelmann JC, Saugspier M, et al. Increased expression of c-jun in nonalcoholic fatty liver disease. Laboratory investigation; a journal of technical methods and pathology 2014;94: Hellerbrand C, Jobin C, Licato LL, et al. Cytokines induce NF-kappaB in activated but not in quiescent rat hepatic stellate cells. The American journal of physiology 1998;275:G

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed. Supplemental Figure 1. DLKI-DIO3 mirna/mrna complementarity. Complementarity between the indicated DLK1-DIO3 cluster mirnas and the UTR of SOX2, SOX9, HIF1A, ZEB1, ZEB2, STAT3 and CDH1with mirsvr and PhastCons