**3. Results**

described by Murphy et al. [51] for DLK1-DIO3 and Fornari et al. [52] for C19MC. Reactions were performed with 30 ng treated DNA using SYBR Green Master Mix (Roche). Quantification was carried out using a standard curve generated using a dilution series of fully methylated with unmethylated DNA (Applied Biosystems). Each sample was analyzed in duplicates, and

Three genomic regions located along each of the clusters were chosen for CN estimations of DLK1-DIO3 and C19MC. Assay qBiomarker Copy Number (Qiagen) was used. Genomic DNA from the stromal cell line HS-5 (CRL-11882) was applied as a calibrator. Analysis was performed with 5 μl of SYBR Green Master Mix, 0.5 μl of respective copy number assay, and 2 ng of genomic

The transient knockdown of SIRT3 was performed in HD-BMMSC using siRNA (Qiagen). The transfections were carried out in 6-well and 24-well plates. For a 24-well plate, 33 nM siRNA was mixed with 6 μl HiPerFect Transfection Reagent in 100 μl serum-free medium and incubated for 10 min at room temperature. The cells were incubated for 48 h and then used for future analyses. For a six-well plate, the cell number was constant, and the reagent volumes

Investigation of ROS amount was carried out using the DCFDA—cellular Reactive Oxygen Species Detection Assay Kit (Abcam) as recommended in the instruction. Analysis was con-

Analysis of mitochondrial membrane potential (ΔΨm) was performed with the Mitochondria Staining Kit (Sigma) using JC-1 dye. Results were analyzed using the ratio of JC-1 aggregates

Proteins from complete cell lysates of BMMSC were detected with a Coomassie (Bradford) Protein Assay Kit (Pierce) and were adjusted with BupH Coating Buffer (Pierce). Analyses were performed according to the commercially available indirect ELISA protocol from Abcam. Detection was performed with 1-Step pNpp-Substrate (Pierce). Absorption was measured at 405 nm. All measurements were performed with three technical replicates. A dilution series of

Statistical analysis was performed using GraphPad Prism 6 software (La Jolla, CA, USA). The data shown represent the mean ± standard error of the mean (SEM). Comparisons of

method.

DNA in a total volume of 10 μl. Relative quantification was achieved by the ΔΔC<sup>t</sup>

**2.9. Determination of mitochondrial membrane potential and reactive oxygen** 

(median value of FL2 channel) to JC-1 monomers (median value of FL21 channel).

complete cell lysates of the HS-5 cell line was used for standard curve generation.

Ct values above 32 were excluded.

**2.8. Transfection of SIRT3 siRNA**

were scaled up accordingly.

**2.11. Statistical analysis**

ducted using the median fluorescence intensity.

**2.10. Indirect enzyme-linked immunosorbent assay (ELISA)**

**species**

**2.7. Copy number (CN) variation analysis**

124 Stromal Cells - Structure, Function, and Therapeutic Implications
