Ephrin/Ephrin Receptor Expression in Ammonia-Treated Rat Astrocytes

1

Supplemental Information

Ephrin/ephrin receptor expression in ammonia-treated rat astrocytes

and in human cerebral cortex in hepatic encephalopathy

Karmela Sobczyk, Markus S Jördens, Ayse Karababa

Boris Görg and Dieter Häussinger

Clinic for Gastroenterology, Hepatology and Infectiology

Heinrich-Heine University, Düsseldorf, Germany

Supplemental Tables

Table 1: Patient demographics, cause of death and etiology of cirrhosis in cirrhotic patients with or without HE. 4 controls, 4 patients with cirrhosis without HE and 4 patients with cirrhosis with HE were obtained from the Australian Brain Donor Programs NSW Tissue Resource Centre.

Table 2: Liver and brain pathology and comorbidities of controls and cirrhotic patients with or without HE.4 controls, 4 patients with cirrhosis without HE and 4 patients with cirrhosis with HE as well as the clinical information and the corresponding pathology were obtained from the Australian Brain Donor Programs NSW Tissue Resource Centre.

Table 3: PCR primer sequences used for realtime-PCR analysis. PCR primer sequences are given in 5´ to 3´ direction. f: forward, r: reverse.

1 Ivanov et al. (2005); 2 Buchert et al. (1999); 3 Biervert et al. (2001); 4 Lai et al. (1999); 5 Carmen Döbele (2011); 6 Zhuang et al. (2010).

Table 4: Effect of methionine sulfoximine, L-NAME and apocynin on EphR/ephrin and GLAST mRNA expression levels in cultured rat astrocytes. Astrocytes were treated withmethionine sulfoximine (MSO, 3mmol/l), L-NAME (1mmol/l) and apocynin (300µmol/l) or were left untreated for 72h before RNA was isolated and ephrin/ephrin receptor (EphR) and GLAST mRNA expression levels were analysed by realtime-PCR. mRNA expression levels in MSO-, L-NAME- or apocynin-treated astrocytes are given relative to untreated controls. *: statistically significantly different compared to untreated controls. n.s.: non statistically significantly different compared to untreated controls.

Supplemental References

1. Ivanov AI, Steiner AA, Scheck AC, Romanovsky AA (2005) Expression of Eph receptors and their ligands, ephrins, during lipopolysaccharide fever in rats. Physiol Genomics21:152–60.

2. Buchert M, Schneider S, Meskenaite V, Adams MT, Canaani E, Baechi T, Moelling K, Hovens CM (1999) The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain. J Cell Biol144:361–71.

3. Biervert C, Horvath E, Fahrig T (2001) Semiquantitative expression analysis of ephrine-receptor tyrosine kinase mRNA’s in a rat model of traumatic brain injury. Neurosci Lett 315:25–8.

4. Lai KO, Ip FC, Ip NY (1999) Identification and characterization of splice variants of ephrin-A3 and ephrin-A5. FEBS Lett458:265–9.

5. Döbele C. Functional Characerisation of members of the microRNA-17-92 cluster in the vascular system (2001), Dissertation, Goethe Universität Frankfurt am Main

6. Zhuang Z, Yang B, Theus MH, Sick JT, Bethea JR, Sick TJ, Liebl DJ (2010)EphrinBs regulate D-serine synthesis and release in astrocytes.J Neurosci30:16015-24.

Supplemental Figure Legend

Supplemental Figure 1:Effect of hypoosmolarity on mRNA expression of ephrin receptor isoforms (A) and ephrins (B) in cultured rat astrocytes. Astrocytes were exposed to normoosmotic (320mosmol/l) or hypoosmotic (205mosmol/l) cell culture media for the indicated period of time before RNA was extracted and mRNA expression levels of various ephrin/ephrin receptor isoforms were analysed by realtime-PCR. mRNA expression levels in astrocytes treated with hypoosmotic cell culture media are given relative to the normoosmotic controls. *: statistically significantly different compared to untreated controls.

Supplemental Figure 2: Effect of NH4Cl on GLAST mRNA expression in cultured astrocytes. Astrocytes were either treated with NH4Cl (5mmol/l) or left untreated for the indicated time period. GLAST mRNA expression was analysed by realtime-PCR and expression level are given relative to the respective control. *: statistically significantly different compared to untreated controls.

Supplemental Figure 3:Effect of NH4Cl on mRNA expression of ephrin receptor isoforms (A) and ephrins (B) in cultured rat astrocytes. Astrocytes were either exposed to NH4Cl (5mmol/l) or left untreated for the indicated period of time before RNA was extracted and ephrin and ephrin receptor (Eph) mRNA expression levels were analysed by realtime-PCR. Ephrin receptor and ephrin mRNA expression levels in NH4Cl (5mmol/l)-treated astrocytes is given relative to the normoosmotic controls. *: statistically significantly different compared to untreated controls.

Supplemental Figure 4: Effect of NH4Cl on FGF receptor 1 expression in cultured astrocytes. Astrocytes were either left untreated or treated with NH4Cl (5mmol/l) for 72h before cells were fixed and FGF receptor 1 (FGFR1) was detected by immunofluorescence analysis. Nuclei were counterstained using Hoechst 34580.