1. Patient
This study was performed with the approval of the Ethics Committee of Chang Zheng Hospital, and informed consent was obtained in accordance with the Declaration of Helsinki. A 70-year-old male patient was diagnosed with stage III of International Staging System (ISS), stage IIIA according to Durie–Salmon (DS) staging system multiple myeloma. Bone marrow aspiration showed that plasma cells accounted for 97.5% of the bone marrow cell. His myeloma cell secreted monoclonal protein of immunoglobulin D, kappa chain. The serum IgG was 3.39 g/L, IgA 0.26 g/L, IgM 0.18 g/L. We used pleural effusion cells at the time of cell collection for this experiment.
2. Cell culture and establishment of cell line.
Pleural effusions specimens were obtained from the patient and mononuclear cells were separated by Ficoll density sedimentation. CD138 + cells were isolated using CD138 magnetic beads and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), penicillin (100 U/mL), and streptomycin (100 µg/mL). They were cultured in an incubator under an atmosphere with 5%CO2 at 37℃. Culture medium was exchanged every 2–3 days to maintain optimal cell growth.
3. Morphology and cytogenetic analysis.
Cytospin preparations were routinely prepared and harshly treated (87 °C for 10 minutes, then cooling to 70 °C) before stained by Giemsa to regularly assess the cell type morphology and the presence of mitoses. Cell morphology was examined using light microscopy.
4. FISH analysis
Interphase fluorescent in situ hybridization (FISH) was performed on CZ2 by using the Fluorescent in situ Hybridization Kit (Cytocell, UK), according to the manufacturer’s instructions. Commercial FISH probes targeting 1q21, del 13q14(RB1 gene), del 17p13(TP53 gene), and t (4;14) were used. At least 200 interphase cells with intact nuclei were evaluated for each probe. Positive nuclear staining in more than 20% of the cells was considered positive.
5. Immunophenotypic analysis by flow cytometry
For immunophenotypic characterization of CZ2 cells, the following antibodies were used: CD1a, CD19, CD20, CD21, CD25, CD3, CD28, CD33, CD38, CD45, CD56, CD117, CD138, CD4, CD13, CD27, CD5, CD8, CD9, CD10, CD24, CD15, CD34, CD44, CD89, CD95, CD98, CD96, CD105, CD184, HLA-DR; cytoplasmic κ and γ light immunoglobulin (Ig) chains.
6. Immunoglobulin secretion
To measure the secretion of free immunoglobulin light chains in serum and culture medium of CZ2, the nephelometric technique was used and performed on a Siemens BNII nephelometer (Siemens Healthcare Ltd.).
7. Virus detection
The presence of Epstein–Barr virus (EBV) was examined using EBV Polymerase Chain Reaction (PCR) Fluorescence Detection Kit (Huirui Biotechnology, Shanghai, China). PCR was carried out using an LightCycler480 II (Roche, USA) for 40 cycles.
8. Western Blotting Analysis
The whole cell protein from CZ2 and other MM cells were collected and resolved in 10% SDS-PAGE gels and then electrotransferred to polyvinylidene difluoride membranes (Millipore, USA). Rabbit polyclonal anti-CKS1B antibody (1:500, Invitrogen), rabbit polyclonal anti-PARP antibody (1:1000, Beyotime), rabbit polyclonal anti-caspase3 antibody (1:1000, Beyotime) were used as primary antibodies while horseradish peroxidase-conjugated goat anti-rabbit IgGs (1:3000, Santa Cruz) was used as secondary antibodies. After treating with the secondary antibodies, protein bands were detected by enhanced chemiluminescence.
9. Cell viability analysis
The MTT assay was used to measure the cell viability of CZ2 and other MM cells. These cells were seeded in 96-well plates and transfected with CKS1B siRNA or control siRNA. After 48 h or 72 h of treatment, 10 µl of the viability reagent MTT was added to each well. The plates were incubated in an incubator at 37℃ for 2 hours. The medium was then discarded and 200 µl DMSO was added to each well. The absorbance at 570 nm was then determined with a microplate reader (Tecan, Spark, Switzerland).