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ORAL PRESENTATION / TAM METİN SÖZLÜ SUNUM

▪ BSO 20 + DOX: Cells were pretreated with 20 µg/ml BSO for 30 min and then treated
with 5 µM doxorubicin for 24 h.

▪ BSO 40 + DOX: Cells were pretreated with 40 µg/ml BSO for 30 min and then treated
with 5 µM doxorubicin for 24 h.

▪

Real-Time PCR- Analyzing gene expression in cell lines: After seeded in a 6-well plate at
2 × 10 cells/ml, the cells were treated with DOX (5 μM) or BSO (10, 20, and 40 μg/ml) +
6
DOX (5 μM), followed by 24 h. The cells were harvested from the wells using a scraper and
then homogenized in a Tissue Lyser II device (Qiagen, Germany) (400 µL Lysis Binding Buffer

solution was added to all groups). Total RNA was extracted from the cells using an RNA
isolation kit (EcoPURE total RNA kit, EcoTech, Türkiye) according to the manufacturer's

instructions. Complementary DNA (cDNA) synthesis was performed using a High-Capacity

cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA). A Veriti 96 Well Thermal
Cycler (Applied Biosystems) was used for synthesis. The resulting cDNA was stored at -20

°C. The experiment was performed using the SYBR Green Master Mix kit to quantify the
mRNA expression levels of NF-κB, TNF-α, and caspase-3. Amplification and quantification

were performed using RT-PCR (StepOnePlus RT-PCR, Applied Biosystems). β-Actin was used
as the reference gene. SYBR Green Gene Expression modifies the PCR program according
®
to the manufacturer’s instructions and initiates the process. All results are expressed as fold-
change in expression relative to groups using the 2 −ΔΔCt method.

Statistical analysis: Statistical analyses were conducted using SPSS software (v23.0, IBM
Corp.), and results are presented as the mean ± standard deviation. Analysis of variance

(ANOVA) was used to examine the data, followed by Duncan's test. Statistical significance

was set at P<0.05.

RESULTS
The cytotoxic effects of varying concentrations of BSO (1-640 µg/mL) on H9c2 cells

were evaluated using the MTT assay over a 24-h period. BSO inhibited the proliferation of
H9c2 cells after ≥80 µg/mL. Analysis of the MTT assay data revealed that the IC50 value for

BSO in H9c2 cells was 147 µg/mL. Consequently, concentrations of 10, 20, and 40 µg/ml
were selected for subsequent real-time PCR analysis.

The chemical composition of (%) the blueberry seed oil is presented in Table 1.

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