Cell cultivation and cell stimulation
To mimic ductal breast carcinoma, the T47D cell line was used. Cells were grown on 80% monolayer in a cell culture bottle and cultivated in Dulbecco's Modified Eagle Medium (DMEM; 3.7 g/L NaHCO3, 4.5 g/L d-glucose, 1.028 g/L stable glutamine, and sodium pyruvate; Biochrom, Berlin, Germany). Then 10% heat-inactivated fetal calf serum (FCS; Biochrom) was added to the medium and incubated with atmospheric concentrations of CO2 of 5% at 37 °C. For further use they were trypsinized and counted.
Preparation of the matcha tea extract (MTE)
Matcha tea was purchased commercially (Houjo Matcha Tea, harvested in Hoshino, Yame prefecture, Japan). For each test, the tea extract preparation was done as described in each test below.
T47D cells were cultivated in a 96-well plate at a density of 10,000 cells per well in 50 µl DMEM with 10% FCS. After 4 h, the medium was replaced with DMEM without FCS. FCS is known to contain substances affecting cell proliferation and maintenance such as growth factors, hormones, vitamins, and transport proteins. By using DMEM without FCS, a possible effect of FCS components on cell proliferation was reduced [33, 34]. Cells were incubated for a further 12 h. 27.1 mg tea extract were dissolved in 100 µl pure ethanol and diluted 1:1000 with DMEM without FCS. A control solution was prepared in the same way without adding MTE. Afterwards, different amounts of the MTE solution and DMEM without FCS were added to achieve the desired concentrations (5, 10 and 50 µg/ml), resulting in a total of 100 µl per well. For the control group, instead of the MTE solution, the control solution and DMEM without FCS were added to each well. The wells were then incubated for 72 h. After incubation, the WST-1 reagent (water soluble tetrazolium, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate; Sigma-Aldrich, St. Louis, MO, USA) was added to detect the activity of mitochondrial succinate dehydrogenase cleaving the tetrazolium salt to formazan. Cell viability was measured after incubating for 30 min with the use of a multi-well spectrophotometer (wavelength: 420–480 nm). Three independent measurements with three technical replicates were performed.
T47D cells were incubated on a 12-well plate at a density of 500,000 cells per well for 4 h with 500 µl DMEM with 10% FCS. Then the medium was replaced with 500 µl DMEM without FCS and the cells were incubated for a further 12 h. 27.1 mg tea extract were dissolved in 100 µl pure ethanol and diluted 1:1000 with DMEM without FCS. A control solution was prepared in the same way without adding MTE. Afterwards, different amounts of the MTE solution and DMEM without FCS were added to achieve the desired concentrations (5, 10 and 50 µg/ml), resulting in a total of 500 µl per well. For the control group, instead of the MTE solution, the control solution and DMEM without FCS were added to each well. The wells were then incubated for two hours. This time was selected because changes in mRNA levels are expected within hours after stimulation and the half-life of mRNA also being in the range of hours. Longer incubation times may, therefore, be unsuitable for detection of short-term changes in mRNA levels. The excess liquid was removed. The wells were washed with phosphate-buffered saline (PBS) and RA-1 buffer (Macherey–Nagel, Düren, Germany) was added for cell lysis. For RNA isolation, the NucleoSpinRNAII (Macherey–Nagel, Düren, Germany) was used. For the reverse transcription of RNA, the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used. 10 ng of RNA were added. The temperature protocol phases were 10 min at 25 °C, 2 h at 37 °C, 5 s at 85 °C and stopped with a cooling phase at 4 °C. For the TaqMan® PCR, 96-well plates with 20 µl well volume were used. Each well was filled with 10 µl of TaqMan® Universal PCR Master Mix 2X (Thermo Fisher Scientific), 8 µl of distilled water treated with 0.1% diethyl pyrocarbonate (DEPC), 1 µl of TaqMan® Gene Expression Assay 20X (Thermo Fisher Scientific; target: ACTB, assay ID Hs99999903_m1; target: PPARG, assay ID Hs01115513_m1; sequences of the primers not revealed by manufacturer), and 1 µl of cDNA sample. The PCR assay was performed using the ABI Prism 7500 Fast (Thermo Fisher Scientific). Thermal cycling was started for 20 s at 95 °C and was followed by 40 cycles of amplification at 95 °C for 3 s, and 60 °C for 30 s. To analyze the results, the comparative 2−ΔΔCT method was used . As endogenous control for the ΔCT-values, β-actin was used. Three independent measurements with two technical replicates were performed.
T47D cells were cultivated in a 12-well plate at a density of 500,000 cells per well in 1000 µl DMEM with 10% FCS. After 4 h, the medium was replaced with 1000 µl DMEM without FCS and the cells were incubated for a further 12 h. 20 mg tea extract were dissolved in 100 µl pure ethanol and diluted 1:1000 with DMEM without FCS. The dilution for the control cells was prepared in the same way without the addition of MTE. Afterwards, different amounts of the MTE solution and DMEM without FCS were added to achieve the desired concentrations (5, 10 and 50 µg/ml), resulting in a total of 1000 µl per well. For the control group, instead of the MTE solution, 250 µl control solution and 750 µl DMEM without FCS were added to each well. Cells were incubated for 48 h, then washed with phosphate-buffered saline (PBS). For cell lysis, 200 µl of a buffer solution consisting of a 1:100 of protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA) in RIPA buffer (radioimmunoprecipitation assay buffer; Sigma-Aldrich) was added to each well before incubating for 30 min at 4 °C. After centrifuging the lysates, a Bradford protein assay of the supernatant was performed. With western blotting, the proteins were separated depending on their molecular weight with the use of SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). After blocking the PVDF membrane for 1 h in a receptacle containing a casein solution 1× (Vector Laboratories, Burlingame, CA, USA) to prevent a nonspecific binding of the antibodies, the primary antibodies anti-β-actin (clone AC-15, mouse IgG; Sigma-Aldrich Co., St. Louis, Missouri, USA) and anti-PPARγ (polyclonal IgG, rabbit, Abcam, Cambridge, UK) were diluted in a 1×casein solution and afterwards laid onto the membrane for 16 h at 4 °C. After washing the membranes with tris-buffered saline (TBST), membranes were incubated with biotinylated anti-rabbit IgG antibody and ABC-AmP reagent (VECTASTAIN ABC-AmP Kit for rabbit IgG, Vector Laboratories) according to the manufacturer’s protocol. Specific bands on the membrane were visualized using the BCIP/NBT chromogenic substrate (Vectastain ABC-AmP Kit, Vector Laboratories), detected with the Bio-Rad Universal Hood II (Bio-Rad Laboratories, Hercules, CA, USA) and the specific color densities of the bands were quantified with the Bio-Rad Quantity One software (Bio-Rad Laboratories) (Fig. 1). For statistical evaluation, the color intensity of PPARγ bands clustered pixels were set in relation to the β-actin bands clustered pixels. The western blots were repeated independently five times.
The statistical programming environment R, version 4.0.2 , was used for processing and statistical analysis of the collected data. Findings with p values < 0.05 were considered significant. Shapiro–Wilk tests were used to check normality of distributions. Adapted to the specific design of experiment we applied different statistical tests to compare stimulated and control samples. The paired t test was used for the WST-1 and PCR assay. To account for repeated measurements in several Western-blot analyses we fitted a linear random effects model.