Fig. 3

Targeted knocking down of RAGE restricted autophagy which was associated with the phosphorylation of erk and mTOR. A (a-1) Both Jurkat and HL-60 cells was transfected with shRNA-RAGE and shRNA-NC for 2 weeks. Then cells were subjected to western blotting to verify the knock down of RAGE protein. (a-2) Targeted knocking down of RAGE decreased autophagy that extracellular HMGB1 induced in leukemia cells. Cells that stably transfected with shRNA-NC and shRNA-RAGE were pretreated with 50 ng/mL rHMGB1 for 24 h, then cells were co-cultured with 0.4 μM ADM for another 24 h. Western blotting was used to detect the alternation of autophagy. (a-3) Was the diagram of the quantitative data of LC3II/I in Jurkat and HL-60 cells respectively. (a-4) Both leukemia cells were pretreated with or without rHMGB1 for 24 h, then ADM (0.4 μM) was applied. Cell Counting Kit-8 was used to assess the cell viability. (a-5) Leukemia cells were treated with ADM (0.4 μM) with or without FPS-ZM1 for 24 h, Western blotting was used to detect the alternation of autophagy. B (b-1) Leukemia cells that transfected with shRNA-NC were treated with a small inhibitor of ERK2/1, 10 μM PD98059 together with 50 ng/mL rHMGB1 for 12 h, then 0.4 μM ADM was added into cells. Western blotting was used to detect the change of autophagy and the phosphorylation of ERK and mTOR. (b-2) Showed the quantitative data of P62 and LC3II/I of leukemia cells. C Cells that stably transfected with shRNA-RAGE were pretreated with the small inhibitor of mTOR, Rapamycin (100 nM) for 6 h, following the treatment of ADM (0.4 μM). Cells were subjected to western blotting to assess the level of autophagy and the phosphorylation of mTOR. (c-2) Presented the quantitative data of P62 and LC3II/I of leukemia cells. GAPDH was used as loading control. D Both Jurkat and HL-60 cells that transfected with shRNA-NC and shRNA-RAGE were transfected with mRFP-EGFP-LC3 virus. In the subsequent experiment, cells were pretreated with 50 ng/mL rHMGB1 for 24 h and then 0.4 μM ADM was added for another 24 h. Confocal microscopic analysis is shown with 630× magnification. Data are the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, compared with the untreated group