Figure 2.
Restoration of CTBP2 elicits antimyeloma activity in vitro and in vivo. HMCLs were transduced with GFP empty vector control (EV) or CTBP2-overexpressing vector (CTBP2). Three days after transduction was assigned as day 0 for subsequent functional studies. (A) Stable overexpression of CTBP2 was confirmed using immunoblotting. β-actin served as the internal control. (B) Immunofluorescence study of transduced HMCLs showing nuclear sublocalization of GFP-tagged CTBP2 protein (red) and counterstained with DAPI. Original magnification, 100×. (C) Analysis of cell proliferation using WST-1 analysis at the indicated time points. Cell growth relative to day 0 is presented. (D) Cells were seeded into semisolid methylcellulose medium and cultured for 7 days. Colony-forming ability relative to EV was determined (upper). Representative images of colony morphology under light microscope (lower). (E) Cell cycle distribution in CTBP2-transduced MM.1S and NCI-H929 cells was assessed by propidium iodide (PI) staining. Representative images of cell cycle distribution (left) and statistical analysis of percentages of cells in the Sub G1, G0/G1, S, and G2/M phases (right) are shown. (F) The percentage of cell death in MM.1S (top) and NCI-H929 (bottom) was measured on day 4 using annexin V staining assay. In panels C-F, results are expressed as mean ± standard deviation (SD) of triplicate measurements from at least 3 independent experiments. (G-K) NCI-H929 cells transduced with EV or CTBP2 were injected IV into NSG mice. (G) Bioluminescence imaging (BLI) was performed at the indicated time points and was adjusted to the same scale. (H) Myeloma burden as quantified by BLI in radiance. (I) Survival analysis using the log-rank test. (J) Detection of myeloma load 35 to 41 days after transplantation. The level of myeloma cells in xenografted animals was evaluated by hCD138 and hCD38 staining. (K) Presence of extramedullary plasmacytomas (EMD) and an enlarged kidney found during harvesting. Tumor images and quantification of tumor weight are shown.∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; ns, not significant.

Restoration of CTBP2 elicits antimyeloma activity in vitro and in vivo. HMCLs were transduced with GFP empty vector control (EV) or CTBP2-overexpressing vector (CTBP2). Three days after transduction was assigned as day 0 for subsequent functional studies. (A) Stable overexpression of CTBP2 was confirmed using immunoblotting. β-actin served as the internal control. (B) Immunofluorescence study of transduced HMCLs showing nuclear sublocalization of GFP-tagged CTBP2 protein (red) and counterstained with DAPI. Original magnification, 100×. (C) Analysis of cell proliferation using WST-1 analysis at the indicated time points. Cell growth relative to day 0 is presented. (D) Cells were seeded into semisolid methylcellulose medium and cultured for 7 days. Colony-forming ability relative to EV was determined (upper). Representative images of colony morphology under light microscope (lower). (E) Cell cycle distribution in CTBP2-transduced MM.1S and NCI-H929 cells was assessed by propidium iodide (PI) staining. Representative images of cell cycle distribution (left) and statistical analysis of percentages of cells in the Sub G1, G0/G1, S, and G2/M phases (right) are shown. (F) The percentage of cell death in MM.1S (top) and NCI-H929 (bottom) was measured on day 4 using annexin V staining assay. In panels C-F, results are expressed as mean ± standard deviation (SD) of triplicate measurements from at least 3 independent experiments. (G-K) NCI-H929 cells transduced with EV or CTBP2 were injected IV into NSG mice. (G) Bioluminescence imaging (BLI) was performed at the indicated time points and was adjusted to the same scale. (H) Myeloma burden as quantified by BLI in radiance. (I) Survival analysis using the log-rank test. (J) Detection of myeloma load 35 to 41 days after transplantation. The level of myeloma cells in xenografted animals was evaluated by hCD138 and hCD38 staining. (K) Presence of extramedullary plasmacytomas (EMD) and an enlarged kidney found during harvesting. Tumor images and quantification of tumor weight are shown.∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; ns, not significant.

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