Figure 4.

Lycorine mediated proteasomal degradation of HMGB1 inhibits the dissociation of Bcl-2 from Beclin-1. (A) Release of HMGB1 upon lycorine treatment was analyzed. The amount of HMGB1 was checked in the culture medium of ANBL6 and ARP-1 cells after incubation with or without lycorine for 12 and 24 h by Western blotting. (B) Subcellular localization of HMGB1 was observed under confocal microscope. ANBL6 and ARP-1 cells were treated with or without lycorine for 24 h and then immunostained with HMGB1-specific antibody/Cy3 secondary antibody (shown in red). Nuclei were stained with DAPI (blue). Images were acquired digitally by FV1000-X81 confocal microscope (Olympus, Japan) with 60× magnification. (C) Subcellular localization of HMGB1 was checked by Western blotting. Cytoplasmic and nuclear extracts of the harvested ANBL6 and ARP-1 cells after 24 h treatment with increasing doses of lycorine were prepared and subjected to Western blotting for the detection of HMGB1. Antibodies against α-tubulin and RCC1 were used to determine the purity of the cytoplasmic and nuclear fractions, respectively. (D) Total RNA was prepared from ANBL6 and ARP-1 cells 24 h after lycorine treatment and the level of HMGB1 mRNA was measured by quantitative RT-PCR. (E) Western blotting analysis of total cell lysate prepared from cells treated with 200 μg/ml CHX in the presence or absence of lycorine for the indicated time. The signal intensity from HMGB1 blot was normalized to GAPDH and plotted against the CHX incubation time. (F) Cells were incubated for 18 h with or without lycorine, followed by 6 h with 10 or 15 µM MG-132, and the lysate was used to detect HMGB1. GAPDH was used as a loading control. Densitometry analysis of HMGB1 intensity was performed using ImageJ software and normalized with loading control (HMGB1/GAPDH). (G) Quantitative Co-IP was adopted to investigate the interaction of HMGB1 with ubiquitin. Co-IP was performed using an anti-HMGB1 antibody in lysate from cells treated with or without lycorine and subjected for Western blotting. The blot was then probed with an anti-ubiquitin antibody. (H) Effect of different concentration of lycorine on MEK-ERK pathway. Total protein lysates were analyzed by immunoblotting using the indicated antibodies. Levels of total MEK, ERK 1/2 and Bcl2 were normalized for equal loading to detect their activated phosphorylation state. (I) Association of Bcl-2 with Beclin-1 was analyzed by Quantitative Co-IP. After immunoprecipitation using Beclin-1 antibody, sample was blotted and probed with Bcl-2 antibody.