Fig 1.
LSS induced changes of anti-/pro- inflammatory factors.
(A) mRNA expression of IL-6, VCAM-1 and eNOS after LSS was applied for 0, 30 and 120 minutes. (B-G) Ivabradine reverses LSS caused mRNA alteration of IL-6, VCAM-1 and eNOS. All experiments above were repeated three times.
Fig 2.
Immunofluorescence of IL-6, VCAM-1 and eNOS-Thr495 generated by LSS.
(A-F) Representative images of immunofluorescence. Positive cells in bright green were counted for five different visual fields. (G-L) Bar diagram showing quantitative data of positive cells. *p<0.05 versus LSS 0 minute, # p<0.05 versus LSS 30 or 120 minutes.
Fig 3.
Fluorescence microscopy of ROS-positive cells stained with DHE and DCF and nucleus stained with DAPI.
(A, E) DHE-positive cells in red were counted for five different visual fields. (B, F) DCF-positive cells in bright green were counted for five different visual fields. (C, G) Bar diagram showing quantitative data of DHE-positive cells. (D, H) Bar diagram showing quantitative data of DCF-positive cells. *p<0.05 versus LSS 0 minute, # p<0.05 versus LSS 30 or 120 minutes.
Fig 4.
Effect of LSS on eNOS and mTOR pathway.
(A) Representative western blot images of eNOS-Thr495, p70S6K-Thr389, S6RP-Ser235/236, raptor-Ser792, rictor-Thr1135, Akt-Ser473 and GAPDH that exposed to LSS for time periods. (B-G) Line charts showing intensity of western blot images, all data were normalized by GAPDH. *p<0.05 versus LSS 0 minute.
Fig 5.
Effect of ivabradine on LSS induced changes of eNOS and mTOR pathway.
(A) ECs were exposed to LSS for 30 minutes after pre-treated with ivabradine, protein phosphorylation was compared in cells exposed to LSS with or without pre-treatment of ivabradine. (B-G) Bar diagram showing intensity data of western blot images, all data were normalized by GAPDH. (H) mTORC2 pathway expression in cells exposed to LSS for 120 minutes after pre-treated with ivabradine. (I-K) Bar diagram showing intensity data of western blot images, all data were normalized by GAPDH. *p<0.05 versus LSS 0 minute, # p<0.05 versus LSS 30 or 120 minutes.
Fig 6.
Effect of LY294002 on eNOS and mTOR pathway.
(A) LY294002 was applied before ivabradine pre-treatment and LSS exposure, protein phosphorylation was compared in cells exposed to LSS for 30 minutes with or without ivabradine pre-treatment. (B-F) Bar diagram showing intensity data of western blot images, all data were normalized by GAPDH. *p<0.05 versus LSS 0 minute, # p<0.05 versus LSS 30 minutes, $p<0.05 versus Iva 30 minutes.
Fig 7.
Effect of LY294002 on expression of anti-/pro- inflammatory factors and ROS generation.
(A-C) mRNA expression of IL-6, VCAM-1 and eNOS after LSS was applied with or without LY294002 pre-treatment compared with ivabradine pre-treated cells. (D-F) Bar diagram showing quantitative data of immunofluorescence positive cells. Cells in bright green were counted for five different visual fields. (G-I) Immunofluorescence of IL-6, VCAM-1 and eNOS-Thr495 generated by LSS. (J-K) LY294002 was applied before ivabradine pre-treatment and LSS exposure, ROS generation was compared within groups. (L-M) Bar diagram showing quantitative data of ROS-positive cells. *p<0.05 versus LSS 0 minute, # p<0.05 versus LSS 30 minutes, $p<0.05 versus Iva 30 minutes.