Figure 3.
In vitro activity of MIR17PTi in HMCLs and primary MM (pMM) cells. (A) Dose-dependent activity of MIR17PTi (6-day time point) in HMCLs (n = 10) as assessed by CCK-8 proliferation assay. Live cells are represented as compared (percentage) with untreated controls. (B) Time-dependent activity of MIR17PTi (1 µM) in AMO1, as assessed by qRT-PCR analysis of pri-mir-17-92 (left) and miR-17-92s (middle). The results shown are average pri-mir-17-92 or miRNA expression levels after normalization with glyceraldehyde-3-phosphate dehydrogenase or RNU44 and ΔΔCt calculation (expressed as percentage). Time-dependent activity of MIR17PTi (1 µM) in AMO1, as assessed by CCK-8 assay (right pane). (C) Cell-cycle analysis of AMO1 exposed for 6 days to MIR17PTi (1 µM) or sc-NC (1 µM). (D) Flow cytometric analysis of senescent AMO1 cells (β-galactosidase positive) exposed for 6 days to MIR17PTi (1 µM) o scr-NC (1 µM). (E) Methylcellulose clonogenic assay of AMO1 exposed to MIR17PTi (1 µM) o scr-NC (1 µM; 2-week time point). (F) CCK-8 proliferation assay of HMCLs sensitive (MM.1S, U266, AMO1) or resistant (MM.1R, LR7, ABZB, ACFZ) to conventional anti-MM agents (dexamethasone, melphalan, bortezomib, carfilzomib) exposed to different concentrations of MIR17PTi for 6 days. (G) CCK-8 assay of human bone marrow (BM) stromal cells (hBMSCs), AMO1, or AMO1 cocultured with hBMSCs after 6 days of exposure to indicated concentrations of MIR17PTi. (H) Table showing combination indexes resulting from combinatorial treatments of AMO1 with MIR17PTi and dexamethasone or melphalan or bortezomib (6-day time point). (I) Flow cytometric analysis of 7-AAD–stained pMM cells (n = 13) exposed for 8 days to MIR17PTi or scr-NC (2.5 µM). pMM cells were cultured adherent to GFP+ HS-5 stromal cells (patients 1-3; patients with intramedullary disease) or physically separated from HS-5 by membranes (patients 4-11; patients with intramedullary disease) or alone (patients 12-13; patients with extramedullary disease at relapse [secondary plasma-cell leukemia (sPCL)]). Lower panel (within the frame) shows median activity of MIR17PTi in sensitive pMM cells (n = 10) from the left panel. *P < .05.

In vitro activity of MIR17PTi in HMCLs and primary MM (pMM) cells. (A) Dose-dependent activity of MIR17PTi (6-day time point) in HMCLs (n = 10) as assessed by CCK-8 proliferation assay. Live cells are represented as compared (percentage) with untreated controls. (B) Time-dependent activity of MIR17PTi (1 µM) in AMO1, as assessed by qRT-PCR analysis of pri-mir-17-92 (left) and miR-17-92s (middle). The results shown are average pri-mir-17-92 or miRNA expression levels after normalization with glyceraldehyde-3-phosphate dehydrogenase or RNU44 and ΔΔCt calculation (expressed as percentage). Time-dependent activity of MIR17PTi (1 µM) in AMO1, as assessed by CCK-8 assay (right pane). (C) Cell-cycle analysis of AMO1 exposed for 6 days to MIR17PTi (1 µM) or sc-NC (1 µM). (D) Flow cytometric analysis of senescent AMO1 cells (β-galactosidase positive) exposed for 6 days to MIR17PTi (1 µM) o scr-NC (1 µM). (E) Methylcellulose clonogenic assay of AMO1 exposed to MIR17PTi (1 µM) o scr-NC (1 µM; 2-week time point). (F) CCK-8 proliferation assay of HMCLs sensitive (MM.1S, U266, AMO1) or resistant (MM.1R, LR7, ABZB, ACFZ) to conventional anti-MM agents (dexamethasone, melphalan, bortezomib, carfilzomib) exposed to different concentrations of MIR17PTi for 6 days. (G) CCK-8 assay of human bone marrow (BM) stromal cells (hBMSCs), AMO1, or AMO1 cocultured with hBMSCs after 6 days of exposure to indicated concentrations of MIR17PTi. (H) Table showing combination indexes resulting from combinatorial treatments of AMO1 with MIR17PTi and dexamethasone or melphalan or bortezomib (6-day time point). (I) Flow cytometric analysis of 7-AAD–stained pMM cells (n = 13) exposed for 8 days to MIR17PTi or scr-NC (2.5 µM). pMM cells were cultured adherent to GFP+ HS-5 stromal cells (patients 1-3; patients with intramedullary disease) or physically separated from HS-5 by membranes (patients 4-11; patients with intramedullary disease) or alone (patients 12-13; patients with extramedullary disease at relapse [secondary plasma-cell leukemia (sPCL)]). Lower panel (within the frame) shows median activity of MIR17PTi in sensitive pMM cells (n = 10) from the left panel. *P < .05.

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