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Figure legends
Fig. 1. Icariin selectively inhibited breast cancer cells proliferationin vitro . (A) Natural resources and chemical structure of icariin. (B, C, D and E) Cell viability analysis of MDA-MB-231, MDA-MB-453, 4T1 and MCF-10A after treatment with indicated concentrations of icariin for 24, 48 and 72 h by MTT assay. (F and G) Colony formation of MDA-MB-231, MDA-MB-453, 4T1 and MCF-10A after treatment with different concentrations of icariin. Bars represent means ± SD of at least three independent experiments; *, P < 0.01, **, P < 0.005 and ***, P < 0.001 in comparison with control group.
Fig.2. Effects of icariin on the intrinsic apoptosis mechanism in breast cancer cells. (A and B) MDA-MB-231, MDA-MB-453, 4T1 and MCF-10A cells were exposed to indicated concentrations of icariin and then analyzed apoptosis by FCM using Annexin V/PI dual-staining assay. (C) Western-blot analysis of apoptotic proteins in MDA-MB-231 cells after treatment with indicated concentrations of icariin. (D) The protein expression ratio of Bax/Bcl-2 in icariin treated MDA-MB-231 cells. (E) Changes of mitochondrial membrane potential (ΔΨm) in MDA-MB-231 cells after treatment of icariin. (F and G) The level of ROS was measured in MDA-MB-231 cell after exposed to icariin with or without pretreatment with NAC (10 μM). (H) Overview of mitochondrial apoptosis triggered by icariin in breast cancer cells. Bars represent means ± SD of at least three independent experiments; *,P < 0.01, **, P < 0.005 and ***,P < 0.001 in comparison with control group.
Fig. 3. Icariin suppressed migration and invasion in breast cancer cells. (A) After treatment with icariin, MDA-MB-231 cell was measured by wound healing, transwell migration and transwell invasion. (B) After treatment with icariin, 4T1 cell was measured by wound healing, transwell migration and transwell invasion. Bars represent means ± SD of at least three independent experiments; *, P< 0.01, **, P < 0.005 and ***, P< 0.001 in comparison with control group.
Fig. 4. Icariin suppressed breast cancer cells migration and invasion via SIRT6/NF-κB/EMT signaling pathway. (A and B) The levels of SIRT6 and acylated H3K9 of icariin treated cells determined by western-blot analysis. (C and D) Expression levels of NF-κB associated proteins of icariin treated cells determined by western-blot analysis. (E and F) Expression levels of EMT associated proteins of icariin treated cells determined by western-blot analysis. (G and H) The levels of acylated H3K9 and p-IκBα of icariin treated cells which was pre-treated with oss-128167 (20 μM) determined by western-blot analysis. (I) Immunofluorescent analysis of nuclear transportation of NF-κB p65 protein in MDA-MB-231 cell. (J) Wound healing, transwell migration and transwell invasion assessment of icariin in MDA-MB-231 cells with or without pre-treatment with oss-128167. Bars represent means ± SD of at least three independent experiments; *, P < 0.01, **,P < 0.005 and ***, P < 0.001 in comparison with control group.
Fig. 5. Transcriptomic analysis of icariin in MDA-MB-231 and MCF-10A cells. (A) Heatmap depiction of differentially expressed genes between different treated groups in MDA-MB-231 and MCF-10A cells. (B and C) KEGG analysis of representative signaling pathways enrichment between different treated groups in MDA-MB-231 and MCF-10A cells respectively. (D) Heatmap depiction of differentially expressed genes of NF-κB and TNF signaling pathways between different treated groups in MDA-MB-231 and MCF-10A cells.
Fig. 6. Icariin inhibited tumor growth and regulated tumor immunosuppressive microenvironment. (A) Representative image of MDA-MB-231 tumors of different groups at the termination of experiment. (B) The MDA-MB-231 tumor growth curves of different groups within the treatment process. (C) MDA-MB-231 tumor weight of different treated groups at the termination of experiment. (D) Variation of mice body weight of different groups within treatment progress. (E) Immunohistochemistry analysis of Ki-67 and cleaved caspase3 of MDA-MB-231 tumor sections after different treatments. (F) Expression level of NF-κB associated proteins of different treated MDA-MB-231 tumor tissues were detected by western-bot. (G) Representative image of 4T1 tumors of different groups at the termination of experiment. (H) The 4T1 tumor growth curves of different groups within the treatment process. (I) 4T1 tumor weight of different treated groups at the termination of experiment. (J) Variation of mice body weight of different groups within treatment progress. (K) Immunohistochemistry analysis of Ki-67 and cleaved caspase3 of 4T1 tumor sections after different treatments. (L) Expression levels of NF-κB associated proteins of different treated 4T1 tumor tissues were detected by western-bot. (M) Changes of proportion of CD4+ and CD8+ T cells in 4T1 tumors after different treatments. (N) Changes of proportion of MDSCs in 4T1 tumors after different treatments. Bars represent means ± SD of at least three independent experiments; *, P < 0.01, **,P < 0.005 and ***, P < 0.001 in comparison with control group.
Fig. 7. Icariin inhibited metastasis in the pulmonary metastatic tumor mouse model of 4T1. (A) Bioluminescence images of mice bearing pulmonary metastasis model of 4T1-luciferase cells after different treatments at determined time points (7, 10, 13, 16 days). (B) Quantitation of bioluminescence signal in mice after different treatments at determined time points. (C) Number of nodules of lungs from different treated groups. (D) Images of in vitro lungs from different treated groups. (E) H&E staining analysis of lung tissues from different treated groups. (F) Overview of pathways for icariin-mediated apoptosis induction and anti-metastasis in breast cancer cells. Icariin induced apoptosis, inhibited metastasis and regulated immunosuppressive microenvironment by SIRT6/NF-κB signaling pathway in triple-negative breast cancer cells. oss-127167, the inhibitor of SIRT6.