Figure 1
Figure 1. C/EBPβ is highly expressed in MM cells. (A top panel) Expression of C/EBPβ in MM cell lines was detected by Western blotting as described in “Western blot analysis.” β-Actin was used as a loading control for all samples. (A bottom panel) Induction of expression of C/EBPβ by IL-6. Cells were cultured in 1% FBS with or without IL-6 for 48 hours, and analyzed for C/EBPβ protein expression. (B) C/EBPβ mRNA expression in MM.1S and H929 MM cell lines, B cells, and nonmalignant plasma cells were analyzed by quantitative RT-PCR. Data were analyzed according to the ΔCT method. The results are expressed as C/EBPβ mRNA expression relative to β-actin. B cells were cultured in a cocktail of CD40 and human IgM for 6 days for the differentiation into plasma cells. Expression of CD19 of B cells as well as CD38 for plasma cells was confirmed by flow cytometry. (C) Mononuclear cells of bone marrow samples of 4 MM patients were collected by Ficoll. CD138+ and CD138− cells from the same patient were separated using magnetic-activated cell sorting beads selection. The total RNA was extracted, subjected to cDNA synthesis, and used for quantitative RT-PCR. Results are depicted as C/EBPβ mRNA fold expression in CD138+ cells compared with CD138− cells. The level of mRNA was normalized to β-actin expression. Expression of C/EBPβ and IRF4 was detected by Western blotting with anti-C/EBPβ and anti-IRF4 in CD138+ cells and CD138− cells from the same patient. β-Actin was used as a loading control. Error bars indicate SD of the mean. (D) Immunohistochemical double staining was performed on paraffin-embedded MM bone marrow trephines to detect C/EBPβ expression in CD138+ cells. Images were obtained using an Olympus 1 X70 microscope equipped with a 20×/0.40 numeric aperture objective lens (Olympus) and were acquired through Magnafire Version 4.1 software (Optronics). Positive staining shows a red nucleus for C/EBPβ and brown membrane for CD138. Positive double staining is indicated in panels Di and ii by arrows, negative double staining is shown in panel Diii. Nonneoplastic plasma cells in palatine tonsils did not show a C/EBPβ expression (Div). Vertical lines have been inserted to indicate a repositioned gel lane.

C/EBPβ is highly expressed in MM cells. (A top panel) Expression of C/EBPβ in MM cell lines was detected by Western blotting as described in “Western blot analysis.” β-Actin was used as a loading control for all samples. (A bottom panel) Induction of expression of C/EBPβ by IL-6. Cells were cultured in 1% FBS with or without IL-6 for 48 hours, and analyzed for C/EBPβ protein expression. (B) C/EBPβ mRNA expression in MM.1S and H929 MM cell lines, B cells, and nonmalignant plasma cells were analyzed by quantitative RT-PCR. Data were analyzed according to the ΔCT method. The results are expressed as C/EBPβ mRNA expression relative to β-actin. B cells were cultured in a cocktail of CD40 and human IgM for 6 days for the differentiation into plasma cells. Expression of CD19 of B cells as well as CD38 for plasma cells was confirmed by flow cytometry. (C) Mononuclear cells of bone marrow samples of 4 MM patients were collected by Ficoll. CD138+ and CD138− cells from the same patient were separated using magnetic-activated cell sorting beads selection. The total RNA was extracted, subjected to cDNA synthesis, and used for quantitative RT-PCR. Results are depicted as C/EBPβ mRNA fold expression in CD138+ cells compared with CD138− cells. The level of mRNA was normalized to β-actin expression. Expression of C/EBPβ and IRF4 was detected by Western blotting with anti-C/EBPβ and anti-IRF4 in CD138+ cells and CD138− cells from the same patient. β-Actin was used as a loading control. Error bars indicate SD of the mean. (D) Immunohistochemical double staining was performed on paraffin-embedded MM bone marrow trephines to detect C/EBPβ expression in CD138+ cells. Images were obtained using an Olympus 1 X70 microscope equipped with a 20×/0.40 numeric aperture objective lens (Olympus) and were acquired through Magnafire Version 4.1 software (Optronics). Positive staining shows a red nucleus for C/EBPβ and brown membrane for CD138. Positive double staining is indicated in panels Di and ii by arrows, negative double staining is shown in panel Diii. Nonneoplastic plasma cells in palatine tonsils did not show a C/EBPβ expression (Div). Vertical lines have been inserted to indicate a repositioned gel lane.

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