Cell culture and transfection
Human DFCs were obtained from ALL Cells. DFCs were cultivated in DMEM (Dulbecco’s Modified Eagle Medium, Sigma), supplemented with 10% fetal bovine serum (Sigma) and Penicillin/Streptomycin (Sigma) as described previously [7]. Medium exchange was done twice a week. The cells were washed with PBS (Sigma) during medium exchange and cell passage. The cell passage was performed with a 1:10 dilution of 2.5% trypsin (Gibco). DFCs were seeded at a density of 5000 cells / cm2. Cells at higher passages before and after the induction of cellular senescence were used for analyzes.
Real-time reverse-transcription (RT) PCR array gene expression analyses
For the evaluation of DNA damage and cellular senescence marker gene expression, the Biorad PrimePCR array (DNA damage - Inhibition of telomerase activity and cellular senescence) was used. Total RNAs from DFCs before (passage 9) after the induction of the cellular senescence (DFC_F: passage 21; DFC_S: passage 22) were reverse transcript with iScriptTM cDNA Synthesis Kit (Biorad). For control cells in standard medium was used at the same time point of cell culture. Results were analyzed with the PrimePCR™ Analysis Software (Biorad) and the output is presented as a Volcano Plot. For comparison of all samples a Clustergram was created. Here, red tiles signify a high gene expression, while black/grey and green tiles show a middle gene expression and a low gene expression, respectively. Black tiles with a cross designate no gene expression.
Real time quantitative polymerase chain reaction (PCR) of telomere length
Genomic DNA (gDNA) concentrations from DFC_F and DFC_S (various passages) were measured with the Nano Drop (Thermo Scientific) after isolation with the QIAamp DNA Mini kit (Qiagen).
The real time PCR-based method for telomere length measurement was described previously [16]. Following primers for telomeres and a single-gene copy gene for control were used:
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Telomer:
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te11b: 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′;
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te12b: 5’GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′.
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Single Copy Gene 36B4u,
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36B4u: 5′-CAGCAAGTGGGAAGGTGTAATCC-3′;
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36B4d: 5′-CCCATTCTATCATCAACGGGTACAA-3′.
The SsoAdvanced™ Universal SYBR® Green Supermix (BioRad) and the StepOnePlusTM Real-Time PCR System (Thermo) were used for gene amplification. 36B4 PCRs were carried out with following protocol: the enzyme was activated at 95 °C for 2 min, followed by 30 cycles of 95 °C for 5 s, 58 °C for 10s, and 72 °C for 40s. Telomere PCRs were carried out with 95 °C for 2 min, followed by 20 cycles of 95 °C for 5 s, 56 °C for 10 s, and 72 °C for 60 s. Telomere PCR results were normalized to single copy gene PCR results. The normalized PCR results of DFC_F were calibrated to the normalized PCR result of a single sample of DFC_S.
Flow cytometry analysis
The number of aneuploid cells (> 4 N) was measured by estimation of the DNA content per cell. For this purpose cells were harvested by trypsin-EDTA treatment, washed with PBS and stained first with 4′,6-Diamidin-2-phenylindol (DAPI) (25 min, 37 °C). Analyses were performed with FACS Canto II and FACSDiva software (Becton Dickinson, Heidelberg, Germany).
Qualitative and quantitative β-galactosidase activity assays
The qualitative activity assay is measured according to the manufacturer’s instructions (Cell Signaling Technology) using X-gal (5-bromo-4-chloro- 3-indolyl β-D-galactoside) staining at pH 6.0. Blue stained cells are visualized by light microscope.
For the quantitation of the senescence associated β-Galactosidase activity we used a modified assay, which is described elsewhere [17]. Briefly, we used the 96-Well Cellular Senescence Assay Kit (Cell Biolabs, Inc.). Here, the activtiy is measured by the rate of conversion of 4-methylumbelliferyl-α-D-galactopyranoside to a fluorescent hydrolysis product 4-methylumbelliferone at pH 6.0. Cells were grown before in 60-mm plates for 1 day (high cell passages) or until confluence (low cell passage). The lysed cells were then used for the β-Galactosidase activity assay and for measuring the DNA concentration by Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo-Fisher). The β-Galactosidase activity reactions were carried out at 37 °C for 1 h. The reaction mixture was read by using a 96-well plate using a plate reader with excitation at 385 nm, emission at 465 nm. The relative β-Galactosidase activity is expressed as: [fluorescence of β-Galactosidase activity] divided by [fluorescence of DNA concentration]. Results were calibrated to the relative activity of DFC_S (relative units).
Western blot
Cytoplasmic proteins were isolated from DFCs before and after cellular senescence with protein isolation buffer (250 μl phosphatase, 100 mM Na3VO4, 137 mM NaCl, 200 mM Tris, 480 mM NaF, 1% NP-40, 10% Glycerol + 1 Protease Inhibitor Cocktail tablet from Roche). Samples were separated by SDS-polyacrylamide gel electrophoresis in 4–15% Mini PROTEAN® TGX Stain- Free™ Protein Gels (BioRad) and blotted to a nitrocellulose membrane. Membranes were blocked with skimmed milk or BSA and incubated with primary antibodies for CDK4, P16, P21, P27, AKT1, E2F1 (Cell Signaling), and P53 (Santa Cruz). Secondary antibodies were used according to manufactures instructions. The detection of antibodies was performed by chemiluminescence with the ChemiDoc Imaging System (BioRad). The staining of total proteins was used as loading control.
Statistics
Statistical analyses were performed using the statistical software SPSS 23 (SPSS Inc., Chicago, IL, USA). Statistical analyses were performed with a one-way ANOVA and a Tukey’s post-hoc test. A p-value < 0,05 was considered as significant.
