Additional file 1 of Chidamide increases the sensitivity of refractory or relapsed acute myeloid leukemia cells to anthracyclines via regulation of the HDAC3 -AKT-P21-CDK2 signaling pathway
Creators: name: Wang, Hao
name: Liu, Yu-chen
name: Zhu, Cheng-ying
name: Yan, Fei
name: Wang, Meng-zhen
name: Chen, Xiao-su
name: Wang, Xiao-kai
name: Pang, Bao-xu
name: Li, Yong-hui
name: Liu, Dai-hong
name: Gao, Chun-ji
name: Liu, Shu-jun
name: Dou, Li-ping
Publisher: name: figshare
Year: 2020
Subjects: subject: Biophysics
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 1: Supplemental Figure 1. Anthracycline-resistant cell lines HL60/ADR cells, K562/A02 cells and THP/ADR cells showed resistance to doxorubicin. Cell viability was examined after treatment with different doses of doxorubicin for 24 h by CCK-8 assay. (A) HL60, (B) HL60/ADR, (C) K562, (D) K562/A02, (E) THP-1, (F) THP-1/ADR. Supplemental Figure 2. Differential gene and protein expression in K562 and K562/A02. (A) RT-PCR analysis showed differential gene expression in K562 and K562/A02. (B) Western blot analysis shows protein expression of differential gene in K562 and K562/A02. Data represent three independent experiments, results are shown in the format, mean ± S.D. (*P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, NS: P &gt; 0.05). Supplemental Figure 3. Chidamide sensitizes anthracycline-resistant cells to anthracycline in vivo. (A) K562 cells (1 × 107 cells) were implanted into NOD/SCID mice. The mice were randomly divided into 4 groups (5 mice in each group). Doxorubicin was injected into the mice at day 5 and day 12 after leukemic cells inoculation. And chidamide was administered 3 times every week from day 6 after leukemic cells inoculation. (B) The volume of each tumor was measured every 3 days. The tumor volume was calculated by the formula (V = 0.5*length*width2). (C) The visual analysis of tumors harvested from mice. (D-E) The measurement of xenograft tumor volume and weight. (F) The mRNA expression levels of HDAC3, AKT, P21 and CDK2 were measured using RT-PCR in tumor sections. Data represent three independent experiments, data are expressed as mean values± S.D. (*P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, NS: P &gt; 0.05). Supplemental Figure 4A-I. Chidamide affected the behavior of anthracycline- resistant cells. (A) CCK-8 assays were used to assess the proliferative abilities of K562/A02 cells. The proportion of G0/G1 phase (B) and S phase (C) were measured in response to incubation with chidamide in K562/A02 cells. The proportion of G0/G1 phase (D) and S phase (E) were measured in THP-1/ADR cells. (F-G) represent apoptosis analysis with flow cytometry in K562/A02 cells and (H-I) in THP-1/ADR cells (Annexin V/PI). Data represent three independent experiments, results are shown in the format, mean ± S.D. (*P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, NS: P &gt; 0.05). Supplemental Figure 4J-W. K562, HL60 and patient-derived anthracycline-sensitive AML cells are sensitive to chidamide monotherapy, and chidamide sensitizes anthracycline-resistant cells to anthracycline in K562/A02 and THP/ADR cells. CCK-8 assays were used to assess the proliferative abilities of K562 (J), HL60 (K), patient-derived cells (L). (M-N) are representatives of flow cytometry (Annexin V/PI) for detection of apoptosis. The apoptotic rate of cells was measured by flow cytometry in K562 (O) and HL60 cells (P). (Q-R) represent cell cycle analysis with flow cytometry in K562 cells and HL60 cells. The proportion of G0/G1 phase and S phase were measured in response to incubation with chidamide in K562 cells (S) and HL60 cells (T). The cell viability of (U) K562/A02 and (V) THP-1/ADR cells treated by combination therapy were measured with CCK-8 assays. (W) HDAC activity of K562/A02 cells was measured after treated with chidamide. Data represent three independent experiments, results are shown in the format, mean ± S.D. (*P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, NS: P &gt; 0.05). Supplemental Figure 5. Molecular mechanisms of chidamide activity in K562/A02 and THP-1/ADR cells. Anthracycline-resistant AML cells were treated with different concentrations of chidamide. Expression levels of HDAC3, AKT, P21 and CDK2 were measured using RT-PCR in K562/A02 cells (A) and THP-1/ADR cells (B). (C) K562/A02 and (D) THP-1/ADR were treated with chidamide and the expression levels of HDAC3, P-AKT, AKT, P21 and CDK2 were measured by Western blot. RT-PCR analysis of HDAC3, AKT, CDK2 and P21 (E) and western blot analysis (F) in K562/ADR cells treated with a combination of chidamide and doxorubicin. The expression of pathway genes was measured by RT-PCR (G) and Western blot (H) in THP-1/ADR cells treated with combination therapy. Data represent three independent experiments, and results are shown in the format, means ± S.D. (*P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001). Figure S6. Transfection with HDAC3 shRNA in K562/A02 cells reduces cell proliferation, increases cell apoptosis, induces cell cycle arrest at G0/G1 phase, and suppresses AKT-P21-CDK2 signaling pathways. (A) CCK-8 assays were used to assess cell proliferation ability. (B) Cell apoptosis was analyzed after HDAC3 knockdown by flow cytometry using Annexin-V/PI staining. (C) is a representative of flow cytometry plot for detection of apoptosis. (D-E) Cell cycles of K562/A02 cells were analyzed by flow cytometry after PI staining. (F) is a representative of flow cytometry for cell cycle detection. (G) Expression levels of HDAC3, AKT, P21 and CDK2 were measured by RT-PCR. (H) Expression levels of HDAC3, P-AKT, AKT, P21 and CDK2 were measured by Western blot. GAPDH was used as an internal control. Data represent three independent experiments, and results are shown in the format, mean ± S.D. (*P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, NS: P &gt; 0.05). Figure S7. AKT inhibitor reduces cell proliferation, increases cell apoptosis, induces cell cycle arrest at G0/G1 phase, and suppresses the expression of HDAC3, CDK2 and P21. K562/A02 cells were incubated with an AKT inhibitor (MK2206-2HCL). (A) CCK-8 assays were used to assess cell proliferation ability. (B) Flow cytometry was used to analyze cell apoptosis. (C) is a representative of flow cytometry plot for detection of apoptosis. (D-E) Cell cycles of K562/A02 cells were analyzed by flow cytometry after PI staining. (F) is a representative of flow cytometry for cell cycle detection. (G) Expression levels of HDAC3, AKT, P21 and CDK2 were measured by RT-PCR. (H) Expression levels of HDAC3, P-AKT, AKT, P21 and CDK2 were measured by Western blot. GAPDH was used as an internal control. Data represent three independent experiments, results are shown in the format, mean ± S.D. (*P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, NS: P &gt; 0.05). Table S1.Comparison of different anthracycline-based chemotherapy regimens.