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. 2016 Sep 24;6(12):2209–2224. doi: 10.7150/thno.15584

Figure 2.

Figure 2

HMGB1 acts as the key mediator of lycorine induced autophagy inhibition. (A) Samples were prepared and separated by 2D gel electrophoresis to identify differentially expressed protein after lycorine treatment (5µM). The gel was stained with Coomassie blue and photographed. Section of the photograph is shown (top). The spot indicated by the red arrow in the top right panel corresponding to the most differentially expressed protein comparing the control (top left panel). HMGB1 was identified by the MASCOT search engine (bottom panel). The MASCOT search engine was used to assess the data from LC-MS/MS to identify the proteins from the UniProt protein database. HMGB1 had the highest hit score (549) among all possible hits contained in the database that matched the peptides from the sample. (B) ARH-77, ANBL6, ARP-1 and MM.1S cells were treated with indicated concentration of lycorine for 24 h and the expression of HMGB1 was analyzed in the cell extract by Western blotting. GAPDH was used as a loading control. The intensity of HMGB1 was determined by densitometry using ImageJ software and normalized with loading control (HMGB1/GAPDH). (C) ARP-1 cells were transfected with blank pCMV (Mock) or HMGB1-pCMV (ovHMGB1) vectors and cultured with or without lycorine for 24 h followed by cell viability analysis using a CCK-8 kit. Data are presented from three independent experiments. (D) ARP-1 cells transfected with blank or HMGB1 vectors were treated with or without lycorine and Western blotting was used to investigate the change in autophagy. GAPDH was used as a loading control. (E) ARP-1 cells were transfected with HMGB1 shRNA or scramble shRNA and cultured in the presence or absence of lycorine for 24 h and cell viability was measure using a CCK-8 kit. Data presented are mean±SD from three independent experiments. (F) Scramble or HMGB1 shRNA transfected ARP-1 cells were cultured for 24 h in the presence or absence of lycorine, the whole cell lysate was prepared and subjected to immunoblotting to detect autophagy. GAPDH was used as an internal control.