*4.4. Western Blotting*

For Western blot analysis the cells were seeded in a total of 5 <sup>×</sup> 106 cells per 25 cm2 cell culture flasks and two selected concentration of the compounds were added. After 48 h incubation the cells were harvested, rinsed with cold PBS, suspended in a lysis buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 1% NP-40 and protease inhibitors set) and incubated for 20 min on ice. The suspensions were centrifuged at 10,000 rpm at 4 ◦C for 12 min. Then, a sodium dodecyl sulfate (SDS) sample buffer was added to clear supernatants, and the samples were boiled at 95 ◦C for 5 min and subjected to SDS-PAGE on 10–15% gel. For the tests, the resolved proteins were transferred to a PVDF membrane (Millipore, Billerica, MA, USA), using Semidry Transfer Cell (Bio-Rad, Hercules, CA, USA). After the transfer, the membrane was blocked overnight with 1% casein in TBS at 4 ◦C, and then incubated with primary antibody (dilution 1:2000) (Santa Cruz Biotechnology, USA) at room temperature for 1 h, followed

by secondary horseradish peroxidase-labelled antibody (Dako, Denmark). The bound antibodies were visualized using ChemiDoc Touch Instruments (BioRad, Hercules, CA, USA). The anti-γH2A.X (ab26350) antibody was from Abcam (Cambridge, UK) while the anti-β actin (C-4) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

#### *4.5. Apoptosis Assays by Flow Cytometry*

After seeding at the same density as for cytotoxicity tests in 96-well plates (TPP, Trasadingen, Switzerland) the cells were incubated for 48 h with two selected concentrations of the tested chalcones (5 and 10 μM). For the phosphatidylserine externalization and membrane integrity test, cells were collected, suspended in a binding buffer and stained with Annexin V-FITC and PI (final PI concentration 1 μg/mL). To evaluate caspases 3/7 and 8 the cells were harvested, washed twice with PBS and stained according to the manufacturer's instructions. Briefly, for active caspase 3/7 detection CellEventCaspase-3/7 Green Detection Reagent was added to the samples. For the detection of active caspase 8, FITC-IETD-fmk was added to the samples and the cells were incubated for 0.5 h. Then, the cells were washed twice and re-suspended in wash buffer. Flow cytometric analysis was immediately performed using a flow cytometer (FACS Calibur; Becton Dickinson, Biosciences, San Jose, CA, USA). CellQuest 3.lf. Software (Becton Dickinson, San Jose, CA, USA) was used for data analysis.

#### *4.6. Statistical Analysis*

All data are shown as means with standard deviations (SD). Statistical differences were analyzed using one-way ANOVA followed by Tukey's multiple comparison test. Statistical analysis was performed with STATISTICA version 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA). The results were considered significant at *p* < 0.05.

#### **5. Conclusions**

Modification of the chemical structure of 2- -hydroxychalcone leads to the formation of a derivatives with various antitumor activity in vitro. Such modification consists of creation the analogues containing a methoxy group instead of a hydroxyl group caused both a reduction and an increase in the antitumor strength of the action depending on the number of groups added and their positions. However, the basic mechanism of action did not change, and all the derivatives obtained exerted antiproliferative and proapoptotic activity and, have the capacity to induce DNA damage, but to varying degrees. The cytotoxic effect of the parent compound and derived derivatives was stronger in relation to cancer cell lines than to normal ones. Further research is needed into the possibility of using chalcones as an adjuvant treatment of canine lymphoma or leukemia.

**Supplementary Materials:** The following are available online. Figure S1: 1H NMR spectral of 2- -hydroxy-2-- methoxychalcone (**1**) (CDCl3, 600 MHz), Figure S2: Part of the 1H NMR spectral 2- -hydroxy-2---methoxychalcone (**1**) (CDCl3, 600 MHz), Figure S3: 13C NMR spectral of 2- -hydroxy-2---methoxychalcone (**1**) (CDCl3, 151 MHz), Figure S4: COSY spectral of 2- -hydroxy-2---methoxychalcone (**1**) (CDCl3, 151 MHz), Figure S5: HSQC spectral of 2- -hydroxy-2---methoxychalcone (**1**) (CDCl3, 151 MHz), Figure S6: 1H NMR spectral of 2- -hydroxy-3-- methoxychalcone (**2**) (CDCl3, 600 MHz), Figure S7: Part of the 1H NMR spectral 2- -hydroxy-3---methoxychalcone (**2**) (CDCl3, 600 MHz), Figure S8: 13C NMR spectral of 2- -hydroxy-3---methoxychalcone (**2**) (CDCl3, 151 MHz), Figure S9: HSQC spectral of 2- -hydroxy-3---methoxychalcone (**2**) (CDCl3, 151 MHz), Figure S10: 1H NMR spectral of 2- -hydroxy-4---methoxychalcone (**3**) (CDCl3, 600 MHz), Figure S11: Part of the 1H NMR spectral 2- -hydroxy-4---methoxychalcone (**3**) (CDCl3, 600 MHz), Figure S12: 13C NMR spectral of 2- -hydroxy-4---methoxychalcone (**3**) (CDCl3, 151 MHz), Figure S13: COSY spectral of 2- -hydroxy-4-- methoxychalcone (**3**) (CDCl3, 151 MHz), Figure S14: HSQC spectral of 2- -hydroxy-4---methoxychalcone (**3**) (CDCl3, 151 MHz), Figure S15: 1H NMR spectral of 2- -hydroxy-3--,4--,5---trimethoxychalcone (**4**) (CDCl3, 600 MHz), Figure S16: Part of the 1H NMR spectral 2- -hydroxy-3--,4--,5---trimethoxychalcone (**4**) (CDCl3, 600 MHz), Figure S17: 13C NMR spectral of 2- -hydroxy-3--,4--,5---trimethoxychalcone (**4**) (CDCl3, 151 MHz), Figure S18: COSY spectral of 2- -hydroxy-3--,4--,5---trimethoxychalcone (**4**) (CDCl3, 151 MHz), Figure S19: HSQC spectral of 2- -hydroxy-3--,4--,5---trimethoxychalcone (**4**) (CDCl3, 151 MHz), Figure S20: 1H NMR spectral of 2- -hydroxy-4- ,6- ,3--,4--,5---pentamethoxychalcone (**5**) (CDCl3, 600 MHz), *Molecules* **2020**, *25*, 4362

Figure S21: Part of the 1H NMR spectral 2- -hydroxy-4- ,6- ,3--,4--,5---pentamethoxychalcone (**5**) (CDCl3, 600 MHz), Figure S22: 13C NMR spectral of 2- -hydroxy-4- ,6- ,3--,4--,5---pentamethoxychalcone (**5**) (CDCl3, 151 MHz), Figure S23: HSQC spectral of 2- -hydroxy-4- ,6- ,3--,4--,5---pentamethoxychalcone (**5**) (CDCl3, 151 MHz), Figure S24 HMBC spectral of 2- -hydroxy-4- ,6- ,3--,4--,5---pentamethoxychalcone (**5**) (CDCl3, 151 MHz), Figure S25: 1H NMR spectral of 2- -hydroxy-2--,5---dimethoxychalcone (**6**) (CDCl3, 600 MHz), Figure S26: Part of the 1H NMR spectral 2- -hydroxy-2--,5---dimethoxychalcone (**6**) (CDCl3, 600 MHz), Figure S27: 13C NMR spectral of 2- -hydroxy-2--,5---dimethoxychalcone (**6**) (CDCl3, 151 MHz), Figure S28: COSY spectral of 2- -hydroxy-2--,5---dimethoxychalcone (**6**) (CDCl3, 151 MHz), Figure S29: HSQC spectral of 2- -hydroxy-2--,5---dimethoxychalcone (**6**) (CDCl3, 151 MHz).

**Author Contributions:** Conceptualization, methodology software validation, formal analysis, data curation, A.P.; investigation, A.P., M.H., B.H.S. and M.Ł.; resources, M.Ł. and E.K.; writing-original draft preparation, A.P. and E.K.; writing—review and editing, T.J.; visualization, M.H. and B.H.S.; supervision, B.O.-M. and T.J.; project administration, A.P.; funding acquisition, A.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This publication was financed under the Leading Research Groups support project from the subsidy increased for the period 2020–2025 in the amount of 2% of the subsidy referred to in Art. 387 (3) of the Law of 20 July 2018 on Higher Education and Science, obtained in 2019.

**Acknowledgments:** We would like to thank B.C. Ruetgen (Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna) for providing CLBL-1 cell line and Y. Fujino and H. Tsujimoto (University of Tokyo, Department of Veterinary Internal Medicine) for providing GL-1 cell line.

**Conflicts of Interest:** The authors disclose nonfinancial or personal relationships with other people or organizations that could influence (bias) their work.
