Supplementary data:

Materials and methods

Cell culture and transfection

Chinese hamster ovary (CHO) cells were maintained in modified alpha essential medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine at 37 °C under 5% CO2 conditions. 24 h before transfection, cells were plated into 6 well plates at 90% confluency. The 1290-bp msr/APJ receptor gene fragment was inserted into the pREN expression vector downstream of the cytomegalovirus promoter and upstream of the internal entry ribosome site of encephalomyocarditis virus and the neomycin gene as previously described (Masri et al., 2002). Protein expression was analyzed by western blotting of cell lysates.

Cell Death ELISA Detection

CHO cells expressing APJ were plated at a density of 10,000 cells /well in 24-well plates for 1 d followed by culture in serum-free medium for 48 h in the absence or presence of 10 pM–10 nM apelin. Procedures for Cell Death ELISA Detection were similar to those specified in the main text. To investigate the effects of inhibitors, cells were pretreated with 10 μM PD98059 (an ERK inhibitor) or LY294002 (a PI3-K inhibitor) for 3 h prior to 48 h of apelin treatment.

Detection of MAPK and PI3-K/Akt activation

CHO cells expressing APJ were first treated with 1 nM of apelin for 0-5 min. Procedures for the western blotting were similar to those specified in the main text.

Results

Expression of APJ in transfected CHO cells

Using western blotting, we detected APJ expression in CHO cells transfected with the 1290-bp msr/APJ receptor gene fragment. There were no bands detected in non transfected CHO cells and CHO cells transfected with empty vector.

Supplementary Fig. S1 Expression of APJ in CHO cells. Representative results of immunoblot analysis using an antibody to APJ. Total cellular protein was subjected to immunoblot analysis. The anti-APJ antibody identified a band at 41 kDa. Shown are representative results. Lane 1, lysate from CHO cells transfected with the 1290-bp msr/APJ receptor gene fragment; lane 2, lysate from CHO cells transfected with empty vector; lane 3, lysate from non transfected CHO cells

Apelin protected CHO cells expressing APJ against serum deprivation-induced apoptosis

Cell Death ELISA assay showed that apoptotic CHO cells expressing APJ at 10 pM (2.67 ± 0.20 ELISA absorbance units), 100 pM (2.28 ± 0.17 ELISA absorbance units), 1 nM (1.58± 0.15 ELISA absorbance units) and 10 nM (1.63 ± 0.14 ELISA absorbance units) of apelin, were less than that of controls (2.89 ± 0.23 ELISA absorbance units, all p < 0.05), reaching the maximal anti-apoptotic effect at 1 nM.

Supplementary Fig. S2 Effect of apelin on apoptosis of CHO cells expressing APJ followed by Cell Death ELISA Detection assay. Cells were exposed to 10 pM – 10 nM apelin in serum-free medium for 48 h. Apoptosis was assessed using a Cell Death Detection kit, and expressed as ELISA absorbance units. The bars represent the mean ± SD (n = 6). Asterisks indicate differences significant at the level of 95% confidence compared to the control.

Apelin activated ERK and PI3-K/Akt signaling pathway in CHO cells expressing APJ

Apelin enhanced the levels of phosphorylated ERK and Akt after 5 min of incubation. These data demonstrated that apelin activated ERK and PI3-K/Akt signaling pathways in CHO cells expressing APJ.

Supplementary Fig. S3 Effects of apelin on ERK and Akt activation in CHO cells expressing APJ. Cell lysates were subjected to immunoblot analysis and incubated with p-ERK, ERK, p-Akt and Akt antibodies. Shown are representative results for cells exposed to 1 nM apelin for 0-5 min

APJ/PI3-K/Akt signaling mediates the anti-apoptotic effect of apelin in CHO cells expressing APJ

We examined whether the apelin-induced activation of the ERK and PI3-K/Akt signaling pathway plays a role in cell apoptosis by the Cell Death ELISA assay. Pretreatment of CHO cells expressing APJ with the PI3-K inhibitor LY294002 abolished the anti-apoptotic effect of apelin, but not that of the ERK inhibitor PD98059. Apelin showed no anti-apoptotic effect in non transfected CHO cells. These data indicate that apelin’s anti-apoptotic effect was mediated by the APJ/PI3-K/Akt pathway.

Supplementary Fig. S4 APJ/ PI3-K/Akt signaling pathways mediate the anti-apoptotic effect of apelin in CHO cells expressing APJ. Cells were incubated with PD98059(10 μM)，or LY294002 (10 μM) for 3 h prior to treatment with 1 nM apelin for 48 h. Cell apoptosis was determined by Cell Death Detection kit, and expressed as ELISA absorbance units. The bars represent the mean ± SD (n = 6). Asterisks indicate differences significant at the level of 95% confidence compared to the apelin-treated control.

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