*4.9. Flow Cytometry Analysis*

After incubating with tangeretin for 24 h, cancer cells were dissociated using 1× trypsin/EDTA. We used a previously described method [36]. In total, 1 <sup>×</sup> 106 cells were cultured with anti-CD44-FITC and anti-CD24-PE antibodies (BD, San Jose, CA, USA) on ice for 30 min. The cancer cells were centrifuged and washed two times with 1× FACS buffer and analyzed on an Accuri C6 (BD, San Jose, CA, USA).

#### *4.10. Gene Expression Analysis*

Total RNA was extracted from cancer cells and purified, and real-time RT-qPCR was performed using a real-time One-step RT-qPCR kit (Enzynomics, Daejeon, Korea). We used a previously described method [37]. The specific primers are listed in Table S1.

#### *4.11. Western Blot Analysis*

Protein samples were extracted from mammospheres and cancer cells. After electrophoresis on a 12% SDS-PAGE gel, proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were blocked in Odyssey blocking buffer at room temperature for 1 h and then incubated overnight with primary antibodies. The antibodies were anti-phospho-Stat3, anti-lamin B (Cell Signaling, Danvers, MA, USA), anti-Stat3, anti-Sox2 and anti-β-actin (Santa Cruz Biotechnology, Dallas, TA, USA). After membranes were washed three times using Tris-buffered saline/Tween 20, all membranes were incubated with IRDye 680RD- and IRDye 800W-labeled secondary antibodies for 1 h at room temperature, and the signal images were determined with an Odyssey CLx (LI-COR, Lincoln, NE, USA).

#### *4.12. Electrophoretic Mobility Shift Assays (EMSAs)*

Nuclear proteins were prepared as described previously [38]. EMSAs were performed with an Odyssey Infrared EMSA kit (LI-COR, Lincoln, NE, USA) according to the manufacturer's instructions. IRDye 700-labeled forward and reverse strands of the Stat3 oligonucleotide were annealed. The IRDye 700-labeled Stat3 oligonucleotide was incubated with nuclear extracts in a final volume of 20 μL at room temperature for 20 min. The samples were electrophoresed on a 6% polyacrylamide nondenaturing gel, and EMSA data were visualized on an ODYSSEY CLx system (LI-COR, Lincoln, NE, USA).

#### *4.13. Xenograft Transplantation*

Twelve female nude mice were injected with two million MDA-MB-231 cells with or without an additional tangeretin (2.5 mg/kg) injection. Tumor volume was estimated for 35 days using the formula (width<sup>2</sup> <sup>×</sup> length)/2. The mouse experiments were performed as described previously [39]. Animal care and animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (JNU-IACUC; Approval Number 2017-003) of Jeju National University. Female nude mice (4 weeks old) were purchased from OrientBio (Daejeon, South Korea) and cultured in mouse facilities for 1 week.

#### *4.14. SiRNA of Stat3*

To examine the inhibitory function of Stat3 on mammosphere formation, breast cancer cells were transfected with siRNAs targeting human Stat3 (Bioneer, Daejeon, South Korea). The Stat3 siRNAs (NM\_1145658) and a scrambled siRNA were purchased from Bioneer (Daejeon Cor., South Korea). For siRNA transfection, cancer cells were cultured and transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The protein levels of Stat3 were determined via immunoblot analysis.

#### *4.15. Statistical Analysis*

All data were analyzed with GraphPad Prism 7.0 software (GraphPad Prism, Inc., San Diego, CA, USA). All data from three independent experiments are reported as the mean ± standard deviation (SD). Data were analyzed using one-way ANOVA. A *p*-value of less than 0.05 was considered significant.

#### **5. Conclusions**

A CSC-inhibiting compound from citrus extracts was purified using silica gel, gel filtration, TLC, and HPLC. The compound was identified as tangeretin. Tangeretin inhibited cell proliferation, CSC formation and tumor growth, and modestly induced apoptosis in CSCs. The size of the CSC subpopulation (CD44+/CD24−) was reduced by tangeretin. Tangeretin reduced the total level and phosphorylated nuclear level of Stat3 Tangeretin decreased the transcript levels of Sox2 and Sox2 protein in mammospheres. Our results in this study show that tangeretin inhibits the Stat3/Sox2

signaling pathway and induces CSC death, indicating that tangeretin may be a potential natural compound targeting breast cancer.

**Supplementary Materials:** The following are available online. Specific primer sequences for real-time RT-PCR are described in Table S1. Isolation and structure analysis of CSC inhibitor are described at Figures S1–S9.

**Author Contributions:** H.S.C., and Y.-C.K. designed this study, participated in all the experiments, and H.S.C., and Y.-C.K. wrote the manuscript. J.-H.K., S.-L.K., R.L., and B.-S.Y., helped design and perform the experiments. D.-S.L. supervised the study. All authors have read and agree to the published version of manuscript.

**Funding:** This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1A6A1A03012862, NRF-2020R1A2C1006316) and NRF-2018R1D1A1B07045261).

**Conflicts of Interest:** There are no conflicts to declare.

#### **References**


39. Kim, S.L.; Choi, H.S.; Kim, J.H.; Jeong, D.K.; Kim, K.S.; Lee, D.S. Dihydrotanshinone-Induced NOX5 Activation Inhibits Breast Cancer Stem Cell through the ROS/Stat3 Signaling Pathway. *Oxid. Med. Cell Longev.* **2019**, *2019*, 9296439. [CrossRef]

**Sample Availability:** Samples of the compounds are available from the authors.

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