Table 1.
Primer sequences for quantitative Real-time PCR.
Fig 1.
Influenza virus A H1N1 infects cultured HPMEC effectively.
Cultured HPMEC were infected with H1N1 or vehicle (CTRL) for 1 hour, and then incubated for different periods. The expression of virus M gene in HPMEC was assessed by qPCR (30 cycles). The expression of M gene increases as culture time prolongs, indicating H1N1 infects cultured HPMEC effectively. *, p<0.05; ***, p<0.001. n = 6, from one of three repeated experiments.
Fig 2.
The expression profile of cultured HPMEC infected with H1N1.
HPMEC were infected with H1N1 for 1 h, then cultured for 24h. The expression of target mRNAs was measured by qPCR. Uninfected cells were used as controls. A. The expression of profile of cytokines, B. chemokines, C. interferons, and D. adhesion molecules. n = 4. ***, p<0.001.
Fig 3.
The S1PR1 agonist CYM5442 inhibits elevated ICAM1 expression in HPMEC infected with H1N1.
HPMEC infected with H1N1 were treated with CYM5442 with different dosages and time periods. The expression of ICAM1 was measured by Western blotting at the end of stimulation. The representative images of one of tripled experiments were illustrated. A. HPMEC express S1PR1, which is upregulated upon H1N1 infection. B. CYM5442 inhibits the upregulation of ICAM1 in HPMEC infected with H1N1 with a dosage-dependent manner. *, p<0.05; **, p<0.01, compared to cells treated with vehicle. C. At the dosage of 2μM, the suppressive effect of CYM5442 on ICAM1expression in infected HPMEC is time-dependent. **, p<0.01, compared to infected cells treated with vehicle.
Fig 4.
The suppressive effects of CYM5442 on infected HPMEC are S1PR1-dependent.
The expression of S1PR1 in HPMEC was knocked in (KI) for overexpression, or knocked down (KD) for loss-of-function assays. HPMEC were infected with H1N1 or vehicle, then treated with CYM5442 or control media. The representative images of Western blotting from one of triplicated experiments are shown. *, p<0.05, compared to infected cells treated with vehicle.
Fig 5.
CYM5442 inhibits the activation of NF-κB by a dosage- and time-dependent manner.
Representative images of one of triplicated experiments are illustrated. A. Infected HPMEC treated with different dosages of CYM5442 for 24 h. B. Infected HPMEC treated with 2 μM of CYM5442 for different time periods. *, p<0.05; **, p<0.01 compared to infected cells treated with vehicle.
Fig 6.
The suppressive effects of CYM5442 on the activation of NF-κB are β-arrestin2-mediated.
Representative images of Western blotting from one of triplicated experiments are illustrated. A. CYM5442 increases the expression of are β-arrestin2 in HPMEC infected with H1N1. *, p<0.05. B. Loss of β-arrestin2 blocked the inhibitory roles of CYM5442 on the activation of NF-κB, and overexpression of β-arrestin2 enhanced these suppressive effects. *, p<0.05; *, p<0.01, compared to infected cells treated with vehicle.
Fig 7.
The model of CYM5442-mediated suppression on ICAM1 in infected HPMEC.