**2.5. Quantitative real-time PCR (qPCR)**

Total RNA was extracted using TRIzol as described previously [46]. RNA was treated with DNase (Ambion) and poly(A)-polymerase (NEB) according to the manufacturer's instructions. About 800 ng of RNA was used for cDNA synthesis with a Transcriptor First Strand cDNA Synthesis Kit (Roche) and 2.5 μl of poly(T)VN adaptor primer (10 pmol) in a 20 μl reaction.

qPCR was performed with the FastStart Universal SYBR Green Master Mix (Roche). Primers were designed for each mRNA target using Primer3, OligoCalc, and OligoIDT. MiRNA detection was conducted using a specific miRNA primer and a universal reverse primer complementary to the adaptor sequence [47]. GAP-DH (for mRNA) and 5.8S rRNA (for miRNA) were chosen as housekeeping genes. QPCR was carried out with the Rotor Gene 6000 Real-Time PCR cycler. Cycling condition comprised 10 min at 95°C, 45 cycles of 15 s at 95°C and 60 s at 59°C, followed by a melting curve analysis from 60 to 98°C, rising by 1°/s. Efficiencies of qPCR were determined using linear regression analysis [48, 49] using LinRegPCR software, and relative quantifications were estimated with the Pfaffl method [50]. Received data were analyzed with the Rotor Gene 6000 software.

#### **2.6. Quantitative methylation-specific PCR (qMSP)**

DNA isolation was performed using Puregene reagents (Qiagen) according to the manufacturer's instructions. Genomic DNA was subjected to bisulfite treatment with the EpiTect Fast Bisulfite Conversion Kit (Qiagen) as recommended in the manual. Primers were used as 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 Ct values above 32 were excluded.

HD-BMMSC with MM-BMMSC were performed using the Mann-Whitney *U* test. The Wilcoxon signed-rank test was used for the analysis of co-cultures. Results were considered

Molecular Aberrations in Bone Marrow Stromal Cells in Multiple Myeloma

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**3.1. MM-BMMSCs are characterized by high senescence state and cell cycle abnormalities**

β-galactosidase staining of HD-BMMSC and MM-BMMSC in passage 4.

Analysis of β-galactosidase activity revealed a significantly higher SAβGalA in MM-BMSC when compared with HD-BMDSC (**Figure 1A**). Since no significant differences in senescent cells between passages 1 and 4 were observed in both MM-BMMSC and HD-BMMSC, we can exclude the effect of cultivation on SAβGalA. These results were confirmed by a histological

**Figure 1.** MM-BMMSC exhibits a higher senescence state and a lower self-renewal capacity than HD-BMMSC. *P* values: \* <0.05; \*\* <0.01; \*\*\* <0.001; and \*\*\*\* <0.0001. All data were analyzed using the Mann-Whitney *U* test and unpaired *t*-test (ELISA analysis). (A) Flow cytometric analysis of SAβGalA. ND-MM-BMMSCs and R-MM-BMMSCs displayed higher activity of SAβGalA in passages 1 and 4 of cell cultures compared to HD-BMMSCs. (B) The colony-forming unit fibroblast (CFU-F) assay was used to study the self-renewal capacity of BMMSC. MM-BMMSC showed a lower self-renewal capacity compared

compared to HD-BMMSC. (D) QPCR analysis displayed decreased cyclin E1, increased cyclin D1 and p21 expression in MM-BMMSC compared to HD-BMMSC. (E) Measurement of the protein level in HD-BMMSC and MM-BMMSC. Cyclin E1 was significantly decreased in MM-BMMSC compared to HD-BMMSC, whereas cyclin D1 and p21 were increased. The protein amount of p16 was slightly reduced in MM-BMMSC compared to HD-BMMSCs. ND-MM-BMMSCs, new diagnosed

/G0

phase in MM-BMMSC

to HD-BMMSC. (C) Cell cycle analysis showed a higher amount of cell in S phase and amount in G1

MM patients; R-MM-BMMSCs, MM patients in relapse; HD-BMMSCs, healthy donor control.

statistically significant when *p* ≤ 0.05.

**3. Results**
