*4.7. In Vivo Anti-Tumor Activity*

Eight-week-old female BALB/c mice were purchased from the laboratory animal center of Jiangsu University. Animal studies were conducted under the guidelines of an approved protocol from the Institutional Animal Care and Use Committee at Jiangsu University (Protocol ID: 2021031202). To establish the tumor-bearing mouse model, 0.2 mL of 4T1 cell suspensions (2 <sup>×</sup> <sup>10</sup><sup>7</sup> cells/mL) was inoculated to the mammary fat pad of BALB/c mice. When the tumor volume reached approximately 50–100 mm<sup>3</sup> , tumor-bearing mice were randomly assigned to six groups of five mice each. Fidaxomicin, DAPT, and 5-fluorouracil were first dissolved in DMSO as stock solutions and then administered after dilution with saline to achieve a final concentration of DMSO of less than 0.5%. Mice were treated with fidaxomicin-1 (50 mg/kg), fidaxomicin-2 (25 mg/kg), fidaxomicin-3 (5 mg/kg), DAPT (25 mg/kg), 5-Fluorouracil (25 mg/kg), and saline. Each formulation was injected intratumorally every other day. The anti-tumor efficiency was evaluated by monitoring the tumor volume. The tumor sizes were measured on two vertical axes using a caliper every other day and calculated as (width<sup>2</sup> <sup>×</sup> length)/2. After 24 days, mice were sacrificed, and tumor tissues were excised and weighted.

The expressions of the Hes5 protein in tumor grafts were examined using Western blot assay. Total cell extracts were also prepared using a Cell Total Protein Extraction Kit (Sangon Biotech, Shanghai, China), as mentioned in the above Western blot method. The primary and secondary antibodies involved were the same as those used in the previous Western blot assay.

#### **5. Conclusions**

In summary, we identified fidaxomicin as a potential RBPJ-specific inhibitor through the use of a drug repurposing strategy. The antitumor activity of fidaxomicin was evaluated both in vitro and in vivo. It was shown that fidaxomicin could effectively inhibit the growth of two different breast cancer cell lines at micromolar concentrations. Furthermore, the administration of fidaxomicin resulted in a practical tumor inhibition in 4T1 tumor-bearing mice. Regarding the possible mechanism of action, the treatment of fidaxomicin lowered the expression of Notch downstream target genes at both transcriptional and translational levels, showing that fidaxomicin might potentially inhibit the formation of the RBPJdependent transcriptional complex. These results suggest that fidaxomicin might have potential for the treatment of RBPJ-dependent cancers. Additionally, this study represented an important step towards the repurposing of this FDA-approved drug for the treatment of breast cancer.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph15050556/s1, Figure S1: The chemical structures of compounds used in this study; Figure S2: Schematic diagram of the interaction between Notch, MAML, DNA, and RBPJ; Table S1: Interactions between human transcription factor RBPJ and NICD; Table S2: Interactions between human transcription factor RBPJ and coactivator MAML; Table S3: Primer sequence.

**Author Contributions:** Conceptualization, M.R. and C.F.; methodology, Y.Z., M.C. and W.Z.; investigation, Y.Z., M.C., L.G. and K.M.; validation, D.W. and W.J.; writing-original draft preparation, Y.Z.; writing-review and editing, M.R., D.W. and C.F.; visualization, M.R., Y.Z. and D.W.; supervision, M.R. and C.F.; project administration, M.R.; funding acquisition, M.R. and C.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported, in part, by the National Natural Science Foundation of China (No. 82074286), Natural Science Foundation of Jiangsu Province (Nos. BK20191428, BK20181445), Six Talent Peak Project from Government of Jiangsu Province (No. SWYY-013), the Science and Technology Innovation Fund of Zhenjiang-International Cooperation Projects (GJ2021012), Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (21KJB350023), and Jiangsu University Foundation (20JDG075).

**Institutional Review Board Statement:** The animal study protocol was approved by the Ethics Committee of Animal Experimentation of Jiangsu University with ethics approval clearance certificate no.2021031202.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

