Immu mount

HUVEC were seeded in a Nunc Lab-Tek II 8-Chamber Slide and treated with compounds for indicated time points. Cells were fixed with 4% paraformaldehyde for 203min at room temperature and then permeabilized with 0.5% Triton X-100 (for protein immunostaining) or 0.2% saponin (for co-staining with filipin) for 103min prior to blocking in a blocking buffer (3% bovine serum albumin (BSA) in PBS containing 0.1% Tween-20) for 13h. Cells were incubated with primary antibodies, including anti-LAMP1, anti-PDI, anti-mTOR and anti-GM130 in the blocking buffer overnight at 4°C, followed by the incubation with secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 647 for 13h at room temperature. The cellular nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific). Cells were washed with PBS, mounted with Immu-mount, and observed under a Carl Zeiss LSM 710 confocal microscope. For the analysis of LDL trafficking, HUVEC were pretreated with DMSO or CEP for 8 h, and then 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate-labeled LDL (DiI-LDL) (Thermo Fisher Scientific) was added to the cells. The incubation was continued for 1 h or 6 h before cells were fixed and co-stained with filipin or primary antibodies against LAMP1, PDI and mTOR.