XN4 induces apoptosis in CML or normal primitive and committed progenitors <i>in vitro</i>.

<p>(A) XN4 induced apoptosis in CML CD34⁺CD38^—primitive and CML CD34⁺CD38⁺-committed progenitors. CML CD34⁺CD38^- primitive and CD34⁺CD38⁺ committed progenitors were exposed to XN4 at the indicated concentrations for 48 h. (B) Apoptosis in CML primitive and committed progenitors was analyzed by FACS as the percentage of cells positively labeled with Annexin V-APC (** <i>p<</i>0.01 vs control group, n = 3). (C) Apoptosis in normal primitive and committed progenitors from normal bone marrow was analyzed by FACS as the percentage of cells positively labeled with Annexin V-APC (n = 3).</p

The effect of XN4 on DSBs of DNA damage.

<p>(A) Representation of the fluorescence shift of rH2AX and cleaved PARP. K562 and K562/G01 cells were treated with XN4 for 12 h, 5 mM NAC for 12 h, or 5 mM NAC for 1 h followed by 6 μM XN4 for 12 h. After the treatment, the cells were washed, and then incubated with Alexa Fluor 647-conjugated mouse anti-H2AX (pS139) antibody and 5μl of a PE-conjugated anti-cleaved PARP (Asp214) antibody for 20 minutes at room temperature. The cells were washed once more before flow cytometry. (B, C) Quantification of rH2AX-positive ratio. The data are expressed as the mean±SD (*** <i>p<</i>0.001, ** <i>p<</i>0.01 vs control group, <i>p</i> = NS no significance, n = 3). (D) Representation of DSBs by neutral comet assay. Cells were collected after XN4 treatment for 12 h and processed for neutral comet assay using a CometAssay kit. Approximately 100 nucleus images were captured for each slide and processed by a Zeiss Axio Observer.Z1 microscope. The tail moments from the cells were measured by the TriTek CometScore software. (E) Quantification of the average tail moments (mean±SD) from three independent experiments are presented (** <i>p<</i>0.01 vs control group).</p

Effects of XN4 on apoptosis induction in CML cells.

<p>K562 and K562/G01 cells were incubated 6 μM XN4 or 5 mM NAC for 48 h, and 5 mM NAC pre-treated for 1 h then added 6μM XN4 for 48 h. The cells were harvested, then double-stained with PI and Annexin V-FITC, and subsequently analyzed by flow cytometry. All tests were performed three times, and the data are expressed as the mean±SD (** <i>p<</i>0.01, * <i>p<</i>0.05 vs control group, # <i>p<</i>0.01 XN4 group vs NAC+XN4 group, n = 3).</p

The primer sequences.

<p>The primer sequences.</p

Effects of XN4 on intracellular ROS levels and expression of <i>Nox</i> genes in CML cells.

<p>The fluorescence of DCF shifted after XN4 treatment for 12 h (A, C, D) or 24 h (B, E, F), with or without 5 mM NAC pre-treatment for 1 h. Quantification of DCF intensity fold-change after XN4 treatment. All data are expressed as the mean±SD. (** <i>p<</i>0.01 vs control group, # <i>p<</i>0.01 XN4 group vs NAC+XN4 group, n = 3). (G) The levels of <i>Nox1</i>, <i>Nox2</i>, <i>Nox3</i>, <i>Nox4</i>, and <i>Nox5</i> mRNA were determined using real-time quantitative RT-PCR. The data are presented as fold-changes, which are normalized to <i>GAPDH</i> expression and expressed as the mean±SD. (** <i>p<</i>0.01 vs control group, # <i>p<</i>0.01 XN4 group vs NAC+XN4 group, n = 3).</p

The proliferation inhibition effects of XN4 in K562 and K562/G01 cells.

<p>(A) The chemical structure of XN4. The molecular weight of XN4 is 490 Da. (B) K562 and K562/G01 cells were incubated with XN4 or Nov for 48 h. The cell viability was evaluated by MTT. The data were plotted as a function of percent cell viability based on controls vs. drug concentrations. All studies were performed in triplicate. The concentration of drug at which cell growth was inhibited by 50% (IC₅₀) was estimated using the GraphPad Prism5 software.</p

Effects of XN4 on cell cycle arrest in CML cells.

<p>(A) Representation of apoptosis ratio after XN4 treatment for 12 h. (B) Effects of XN4 on the activation of DNA-damage-sensing kinases in CML cells. K562 and K562/G01 cells were treated with 6 μM XN4 for 12 h, with or without NAC pre-treatment. The data demonstrate that XN4 increased the phosphorylation level of rH2AX and ATM, but decreased CDK2 and CyclinE1; β-Actin served as the protein-loading control. 1. control; 2. XN4 6 μM; 3. NAC 5 mM; 4. NAC 5 mM+XN4 6 μM. (C-E) K562 and K562/G01 cells were cultured in the presence of XN4 for 48 h, with or without pre-treatment with 5 mM NAC for 1 h, harvested, and then stained with PI and subsequently analyzed by flow cytometry with quantitation using the FlowJo software. (F) Representation of the fluorescence shift of BrdU after XN4 treatment, with or without NAC pre-treatment. K562 and K562/G01 cells were treated with 6 μM XN4 for 12 h, 5 mM NAC for 12 h, or 5 mM NAC for 1 h followed by 6 μM XN4 for 12 h. After the treatment, the cells were labeled with BrdU for 30 min, washed, and then incubated with 5μl of an PerCP-Cy5.5-conjugated anti-BrdU antibody for 20 minutes at room temperature. The cells were washed once more before flow cytometry (G) Quantification of the fluorescence shift of BrdU The data are expressed as the mean±SD. (** <i>p<</i>0.01, * <i>p<</i>0.05 vs control group, <i>p</i> = NS no significance, n = 3).</p

Supplementary Figure S4 from Dual Inhibition of Bcr-Abl and Hsp90 by C086 Potently Inhibits the Proliferation of Imatinib-Resistant CML Cells

Supplementary Figure S4. C086 potently inhibits the growth of human leukemia progenitor/stem cells.</p

Supplementary Figure S1 from Dual Inhibition of Bcr-Abl and Hsp90 by C086 Potently Inhibits the Proliferation of Imatinib-Resistant CML Cells

Supplementary Figure S1. C086 induces apoptosis in imatinib-sensitive and -resistant CML cells.</p

Supplementary Figure S3 from Dual Inhibition of Bcr-Abl and Hsp90 by C086 Potently Inhibits the Proliferation of Imatinib-Resistant CML Cells

Supplementary Figure S3. Effects of C086 on cell cycle progression.</p