Additional file 2 of Curcumin derivative C212 inhibits Hsp90 and eliminates both growing and quiescent leukemia cells in deep dormancy
Creators: name: Liu, Bi
name: Shen, Yunzhu
name: Huang, Huafang
name: Croce, Kimiko Della
name: Wu, Min
name: Fan, Yingjuan
name: Liu, Yang
name: Xu, Jianhua
name: Yao, Guang
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 1 : Fig. S1. C212 induces G2/M cell accumulation. (A) Chemical structures of curcumin and C212. (B and C) Growing leukemia cells were treated with C212 at the indicated doses for 24 h and subjected to cell-cycle analysis with PI staining of DNA content (B, K562; C, HL60). Error bar, SEM (n = 2); * p &lt; 0.05, ** p &lt; 0.01 (over control at 0 μM). (D) Cells were treated with C212 as in B and C and subjected to Western blot of Cdc2 and cyclin B1 proteins (left, K562; right, HL60). Fig. S2. C212 binds to Hsp90 and induces proteasome-dependent client protein degradation. (A) Molecular docking simulation of the binding of curcumin, C212, and 17-AAG to Hsp90 at its C-terminus, middle (M-)region, and N-terminus. (B) Growing HL60 cells were pretreated with or without C212 (2.4 μM) for 2 h, followed by co-treatment with or without MG132 (1 μM) for 12 h; whole-cell lysate was then subjected to Western blot. Fig. S3 Growing and serum starvation-induced quiescent HL60 (A) and K562 (H) cells were treated with C212 at the indicated doses for 24 h, followed by Western bolt assay. Protein levels of Hsp90 (B, HL60; I, K562), Hsp70 (C, HL60; J, K562), and Hsp90 client protein p-Erk (D, HL60;K, K562) were normalized to β-actin loading control and plotted against C212 doses. Also shown are p-Erk levels normalized to corresponding Hsp90 levels at tested C212 doses (E, HL60; L, K562). Insets in D-L show the enlarged bar graphs corresponding to Quiescence. Error bar, SEM (n = 2); * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001 (over control at 0 μM). Fig. S4 HCT116 (A) and SW620 (B) cells were serum-starved for 12 and 24 h with EBSS, respectively, to induce quiescence/slow-growth; EdU (10 μM) was added to the medium, and cells were further incubated for 24 h, followed by EdU incorporation assay. Growing: control cells not deprived of serum but otherwise under the same treatment. Error bar, SEM (n = 2). (C and D) Growing and serum starvation-induced quiescent HCT116 (C) and SW620 (D) cells were treated with C212 at the indicated doses for 48 h and subjected to MTS assay. Error bar, SEM (n = 3). (E-N) Growing and serum starvation-induced quiescent cells (E, HCT116; J, SW620) were treated with C212 at the indicated doses for 24 h, followed by Western bolt assay. Protein levels of Hsp90 (F, HCT116; K, SW620), Hsp70 (G, HCT116; L, SW620), and Hsp90 client protein p-Erk (H, HCT116; M, SW620) were normalized to β-actin loading control and plotted against C212 doses. Also shown are p-Erk levels normalized to corresponding Hsp90 levels at tested C212 doses (I, HCT116; N, SW620). Error bar, SEM (n = 2); *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001 (over control at 0 μM). Fig. S5. The effects of 17-AAG versus C212 on leukemia cells. Growing and serum starvation-induced quiescent (as in Fig. 4 a and b) HL60 (A) and K562 (B) cells were treated with 17-AAG at the indicated doses for 48 h, followed by MTS assay. Error bar, SEM (n = 3). Growing HL60 (C-I) and K562 (J-P) cells were treated with C212, 17-AAG, or their combination at the indicated doses for 24 h, followed by Western blot assay. Protein levels of Hsp90 (D-F, HL60; K-M, K562) and client protein p-Erk (G-I, HL60; N-P, K562) were normalized to β-actin loading control and plotted against the treatment doses of C212, 17-AAG, or their combination. Error bar, SEM (n = 2); * p &lt; 0.05, ** p &lt; 0.01, ***p &lt; 0.001 (over control at 0 μM).