Figure 9.

HSP70 promotes the HMGB1b–Beclin 1 autophagy pathway via interaction with Beclin 1. A and B, HMGB1b or HSP70 overexpression up-regulates Beclin 1 protein levels. CIK cells were transfected with HMGB1b-GFP or HSP70-HA for 24 h and subjected to WB analysis with Beclin 1 and β-tubulin Abs. The relative protein level of Beclin 1 was quantified by ImageJ. Error bars indicate S.D. (n = 3); **, p < 0.01. C, knockdown of HSP70 or HMGB1b inhibits H₂O₂-induced protein level of Beclin 1. CIK cells were seeded in 6-well plates and transfected with s1 of HMGB1b, s1 of HSP70, or cotransfected with s1 of HMGB1b and HSP70. Then the cells were treated with H₂O₂ for 24 h. The cell lysate was collected for WB analysis with anti-Beclin 1 Ab. The histograms display the ratios of LC3-II/LC3-I. Error bars indicate S.D.; **, p < 0.01. D, inhibition of HSP70 by VER155008 reduces the H₂O₂-induced Beclin 1 protein level. CIK cells were treated with or without 10 nm VER-155008 for 12 h and then supplied with 0.15 μm H₂O₂ in the medium for another 24 h. The protein levels of Beclin 1 were examined using the anti-Beclin 1 Ab. The relative protein levels of Beclin 1 were quantified by ImageJ. Error bars indicate S.D. (n = 3); **, p < 0.01. E, left panel, HSP70 promotes the interaction between HMGB1b and Beclin 1. HMGB1b-GFP and RFP-HSP70 stably cotransfected cells were pretreated with or without VER-155008 for 12 h. HMGB1b-GFP stably transfected cells were used as a control. All cells were treated with 0.15 mm H₂O₂ for 24 h. IP was conducted with GFP trap beads and IB with Beclin 1 and GFP Abs. WCL was used to analyze the indicated proteins. Right panel, the relative protein levels of Beclin 1, which interacts with HMGB1b, related to the total level of Beclin 1 in WCL, were quantified by ImageJ. Error bars indicate S.D. (n = 3); **, p < 0.01. F, HSP70 co-locates with Beclin 1. CIK cells were cotransfected with GFP-Beclin 1 and RFP-HSP70. Then the transfected cells were treated with rapamycin (Rap) or H₂O₂ for 24 h and subjected to confocal microscope observation. G, HSP70 interacts with Beclin 1. CIK cells were co-transfected with GFP-Beclin 1 and HSP70-HA for 16 h and then treated with or without rapamycin or H₂O₂ for 24 h. Co-IP was performed with anti-HA monoclonal Ab. Mouse IgG was used as a control. WCL was subjected to IB with anti-GFP, anti-HA, and anti-β-tubulin Abs, respectively.