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HEP_24681_sm_suppinfofig1.tif3904KOnline supplemental Fig. 1. Saturation curves of S1P-induced ERK1/2 and AKT activation in primary rat hepatocytes. Primary rat hepatocytes were treated with different amounts of S1P for 20 minutes. The cells were harvested to prepare total cell lysates. The protein levels of phosphorylated ERK1/2 (p-ERK1/2) and AKT (p-AKT) were determined by Western blot analysis and normalized with total ERK1/2 (T-ERK1/2) and total AKT (T-AKT) as described in Materials and Methods. *p<0.05 compared to control group, n=4.
HEP_24681_sm_suppinfofig2.tif6090KOnline supplemental Fig. 2. Expression of S1P1 and S1P2 in hepatocytes. Total RNA was isolated from primary human, rat and mouse hepatocytes. The expression of S1P1 and S1P2 was detected by RT-PCR using specific primers for S1P1 and S1P2. Rat cDNAs of S1P1 and S1P2 were used as positive control (P). The PCR products were purified from DNA agarose gel and further confirmed by DNA sequencing. The representative image of DNA agarose gel was shown.
HEP_24681_sm_suppinfofig3.tif6710KOnline supplemental Fig. 3. Effects of PTX and JTE-013 on activation of ERK1/2 and AKT by S1P in primary rat hepatocytes. (A) Cells were pre-incubated with Pertussis toxin (PTX, 300 ng/ml) for 16 hours or JTE-013 (10 μM) for 30 minutes and then treated with 100 nM S1P for 20 minutes at 37°C. The protein levels of phosphorylated ERK1/2 (p-ERK1/2) and AKT (p-AKT) were determined by Western blot analysis and normalized with total ERK1/2 (T-ERK1/2) and total AKT (T-AKT) as described in Materials and Methods. S1P-induced activation of AKT (A) and ERK1/2 (B) was inhibited by JTE or PTX. *p<0.05 compared to vehicle control; #p<0.05 compared to S1P group, n=4.
HEP_24681_sm_suppinfofig4.tif4934KOnline supplemental Fig.4. Effect of S1P2-shRNA lentivirus on mRNA expression of S1P2 in primary rat hepatocytes. Cells were transduced with control lentivirus or S1P2-shRNA lentivirus for 40 h. Total RNA was isolated. The S1P2 mRNA level was determined by real-time RT-PCR and normalized using ?-actin as internal control. **p<0.01 compared to control lentivirus, n=3.

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