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liv12365-sup-0001-FigS1.pptapplication/ppt2932KFig. S1. Blood biochemical analysis for oxidative stress and CPS-1. Blood samples were collected at different time point after the administration of ammonium chloride, and the levels of MDA (A), SOD (B), GSH-P (C), SOD (D) and CPS-1 (E) were determined by ELISA kits. *indicates P < 0.05.
liv12365-sup-0002-FigS2.pptapplication/ppt692KFig. S2. The effects of pH on hepatic apoptosis can be ruled out in vitro. The human normal hepatic cell line L-02 was chosen to analyse whether the pH has effect on the liver and divided into five groups: A and D, L-02 cells only; B, Cells + medium with HCl (The volume of HCl added to medium was based on the pH value of corresponding group with NH4Cl 0.5 mmol/L; pH value of about 7.3); C, Cells + medium with HCl (The volume of HCl added to medium was based on the pH value of corresponding group with NH4Cl 20 mmol/L; pH value of about 7.1); E, Cells + NH4Cl 0.5 mmol/L; F, Cells + NH4Cl 20 mmol/L. After incubating with NH4Cl or HCl for 24–36 h, all the cells in 12-well chambers were collected and stained with Annexin V-PI to measure apoptosis using flow cytometry.
liv12365-sup-0003-FigS3.pptapplication/ppt230KFig. S3. Validation of the relative efficiency of amplification of the selected genes. Standard curves of seven differentiated expressed genes and two housekeeping genes (Ldha and Hprt 1) was performed using an Applied Biosystems StepOnePlus Real-Time PCR system. Premix Ex Taq was purchased from Takara biology (Otsu, Shiga, Japan). Five different quantities (a dilution series) of the sample were amplified for the validation of the relative efficiency of amplification of the genes.
liv12365-sup-0004-FigS4.pptapplication/ppt38KFig. S4. Representative images of differentiated expressed gene Spp1 verified by RT-PCR. Amplification curve of up-regulated genes Spp1 was performed in hyperammonaemia (hyper-ammonia) and control rats and housekeeping genes Ldha and Hprt 1 were used for expression normalization.
liv12365-sup-0005-FigS5.pptapplication/ppt305KFig. S5. Amplification efficiency of all genes in our study was enough for the 2−△△Ct method. Standard curve of two target genes (Spp1 and Comt) and two housekeeping genes (Ldha and Hprt 1) was performed using an Applied Biosystems StepOnePlus Real-Time PCR system and five different quantities at dilution series of 1:5 (16, 80, 400, 2000, 10 000) of the sample were amplified for the validation of the relative efficiency of amplification of these four genes (Fig. S5A–B). XY scatter plot (Fig. S5C) of △Ct at different diluted points was done using Microsoft Excel, and a trend line and equation were added for genes Spp1 and Comt. The Ct values of relatively low expressed genes (Mx2, Oasli, Foxi2, RGD1563231 and Capza3) at different dilution series (1:3 or 1:2) were shown in Fig. S5D–E. *means quantity of cDNA (without dilution), which came directly from the reverse transcription of RNA, was assumed to be 10000 units, just for convenient calculation.
liv12365-sup-0006-TableS1-S5.docWord document394K

Table S1. Primers and probes used for RT-PCR.

Table S2. The complete microarray dataset of differentially expressed genes in hyperammonaemic rats.

Table S3. The differentially expressed genes associated with cell communication and signal transducer activity.

Table S4. The urea and creatinine levels in hyperammonaemic and control rats.

Table S5. Total RNA Sample 2100 Quality Control (QC) Report.

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