*2.9. Cytokine Array*

The qualitative analysis of human inflammatory cytokines was carried out following the provided instructions using the Multi-analyte ELISArray kit (MEH-003A, QIAGEN, Hilden, Germany). In this assay, each well of the ELISArray plate was loaded with the assay buffer and 50 μL of the cell supernatant. The plate was then allowed to incubate at room temperature for a period of 2 h. Following the incubation, the plate was subjected to a series of washing steps, performed three times using a washing buffer to remove any residual contents from the wells. Subsequently, 100 μL of the detection antibody was added to each well, and the plate was once again incubated at room temperature for 1 h. After this incubation, the plate was washed three times to ensure proper removal of excess detection antibody. Next, 100 μL of avidin-HRP conjugate antibody was introduced to the wells, and the plate was incubated in darkness for 30 min. Following this incubation, the plate underwent four additional washing steps. Subsequently, 100 μL of the developing solution was added to each well, and the plate was placed in darkness for an additional incubation of 15 min. To stop the reaction, an equal volume of stop solution was added to each well. The absorbance of the samples was measured at a wavelength of 450 nm using a microplate reader (Sunrise Technologies).

#### *2.10. Western Blotting*

Cells were washed twice with PBS. To each well, 180 μL of radio-immuno preservation assay (RIPA) buffer (Thermo Scientific) and 1.8 μL of protease and phosphatase inhibitor cocktail (×100) (Thermo Scientific) were added. Centrifugation was performed at 4 ◦C for 20 min at 14,000 rpm. The total protein content was determined using the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific) following the manufacturer's protocol. The sample was mixed with sample loading dye (×5) (Chembio, Medford, NY, USA) and heated at 100 ◦C for 5 min. The mixture was loaded into the wells of a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) setup. The protein was transferred from the gel to a polyvinylidene fluoride (PVDF) membrane filter (Merck Millipore Ltd., Middlesex, MA, USA). The membrane was washed with tris-buffered saline (TBS) containing 50 mM tris-Cl and 150 mM NaCl (pH 7.5) for 5 min. The membrane was blocked with a solution of 5% (*w*/*v*) bovine serum albumin (BSA) (VWR Life Science) at room temperature. The membrane was washed three times with TBS-T (TBS with 0.1% Tween-20), and 10 μL of the primary antibody was diluted in 10 mL of blocking buffer, added to the membrane, and incubated at 4 ◦C for 24 h. The membrane was washed three times with TBS-T. The membrane was exposed to a horseradish peroxidase (HRP) conjugated secondary antibody at room temperature for 1 h. The membrane was washed three times with TBS-T and the expressed protein was visualized using the Western Bright ECL kit (Advansta Inc., San Jose, CA, USA).

#### *2.11. Immunofluorescence*

MDA-MB-231 cells were attached to a 24-well plate containing a coverslip (12 mm, SPL Life Sciences), and various concentrations of mistletoe water extract were added and incubated for 48 h at 37 ◦C in a humidified 5% CO2 incubator (Sanyo). After washing three times with PBS, 500 μL of 4% formaldehyde (pH 7.4) was added and incubated at 37 ◦C for 10 min to fix the cells. After washing three times, 0.1% Triton X-100 was added and incubated for 15 min at room temperature. After washing three times, 500 μL of 2% (*w*/*v*) BSA (VWR Life Science) was added and incubated at room temperature for 1 h. The primary antibody was added and incubated at 4 ◦C for 24 h. After washing three times, the secondary antibody was added and incubated for 45 min at room temperature. After washing three times, DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) for 20 min in the dark and observed with a fluorescence microscope (Nikon).

#### *2.12. Statistical Analysis*

All experiments were conducted in triplicate, and the results are presented as means ± standard deviation (S.D.). Statistical analysis of the data was performed using GraphPad software (version 7.00). Significant differences between groups were determined using a one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison tests for post hoc analysis. Statistical significance was defined as *p* < 0.05, *p* < 0.01, and *p* < 0.001, indicating the level of significance for the observed differences between groups.
