Fig 2: S6K2, but not S6K1, interacts with TRBP during ANG1 treatment of HDLECs. (A) Analysis of TRBP and S6K2 interaction by PLA (red) in HDLECs during 24 h ANG1 stimulation. Nuclei (blue) and F-actin (green) staining are presented in addition (scale bar: 10 µm). PLA events (in red) are indicated with yellow arrows. (B) Bar graph illustrating mean number of TRBP/S6K2 PLA events detected in HDLECs cultured in basal media or following overnight growth factor withdrawal and post 30 min, and 2, 12 and 24 h ANG1 stimulation. (C) Bar graph illustrating mean number of TRBP/S6K2 PLA events detected in HDLECs pre-treated with DMSO or Rapamycin (10 nM) for 2 h prior to 30min and 24 h ANG1 stimulation. (D) Bar graph illustrating mean number of TRBP/S6K1 PLA events detected in HDLECs cultured in Basal media or following overnight growth factor withdrawal and post 30 min, or 12 h ANG1 stimulation. Values presented are means ± SD. P values are shown (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig 3: Endogenous TRBP interacts with S6Ks in primary HDLEC. (A) Analysis of TRBP and S6K2 interaction by PLA (red) in HDLECs growing in full growth factor media. Nuclei (blue) and F-actin (green) staining are also shown (scale bar: 10 µm). PLA events (in red) are indicated with yellow arrows. (B) Analysis of TRBP and DICER interaction by PLA (red) in HDLECs growing in full growth factor media. PLA events (in red) are indicated with yellow arrows. (C) Histogram of TRBP/S6K2 PLA events per cell detected in HDLECs cultured in full growth factor media. (D) Histogram of TRBP/S6K1 PLA events per cell detected in HDLECs cultured in full growth factor media.

Fig 4: Effects of miR-155-5p upregulation on the mTOR signaling pathway.(A) The expression of Rheb, S6K2 and Rictor in rats heart tissue at 15days post-injection was detected by western blot (n = 8 per group). (B) Relative band intensity of Rheb, S6K2 and Rictor in ACM heart tissue (n = 8 per group). (C) Western blot of p-mTOR (ser 2448) in ACM heart tissue (n = 8 per group) transfected with AAV-NC and AAV-miR-155 (n = 8 per group). (D) Relative band intensity of p-mTOR (ser 2448) normalized to mTOR. (E) Real-time quantitative PCR for Rheb, S6K2, and Rictor in heart tissue samples (n = 8 per group). Data are presented as mean ± SD. *p < 0.05; **p < 0.01.

Fig 5: mTOR signaling pathway was activated in cardiac tissues in chronic alcohol intake rats.(A) Western blotting images of Rheb, S6K2 and Rictor in heart tissue samples from model rats after 20 weeks ethanol consumption and the control rats. (B) Relative band intensities of Rheb, S6K2 and Rictor, in heart tissue samples. (C) Western blotting images of p-mTOR (ser 2448) in heart tissue samples. (D) Relative band intensities of p-mTOR (ser 2448). (E) Real-time quantitative PCR for Rheb, S6K2, and Rictor in heart tissue samples (n = 6 per group). Data are presented as mean ± SD. *p < 0.05; **p < 0.01.