Figure 5.
Molecular mechanism underlying MIR17PTi proapoptotic activity. (A) 7-AAD flow cytometric assay of U266MYC−/U266MYC+ after 6 days of treatment with MIR17PTi (2.5 µM) or scr-NC (2.5 µM); western blotting of MYC protein in U266MYC− and U266MYC+ is reported. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as protein loading control (left). 7-AAD flow cytometric assay of P493-6 after 6 days of treatment with MIR17PTi (2.5 µM) or scr-NC (2.5 µM), in presence or absence of doxycycline (dox); western blotting of MYC protein in P493-6 cultured for 2 days with or without doxycycline is reported. GAPDH was used as protein loading control (middle). Trypan blue exclusion staining in MYC-ER human mammary epithelial cells (HMECs) 2 days after transfection with MIR17PTi (50 nM) or scr-NC (50 nM) and cultured with or without tamoxifen (tam) (right). (B) Western blotting of BIM in lysates from healthy donor peripheral blood mononuclear cells (PBMCs; n = 2), Jeko-1 (mantle cell lymphoma; BIM null), Daudi, Raji (Burkitt lymphoma), and indicated HMCLs. GAPDH was used as protein loading control. (C) Western blot analysis of BIM in lysates from pMM cells (patients 12 and 13; extramedullary MM) exposed to MIR17PTi for 6 days at indicated concentrations. GAPDH was used as protein loading control. (D) Western blotting of BIM in AMO1, NCI-H929, or INA-6 exposed for 6 days to MIR17PTi (AMO1, 1 µM; NCI-H929 and INA-6, 2.5 µM) or equimolar scr-NC. GAPDH was used as protein loading control. (E) Western blot analysis of BIM in lysates from AMO1 transfected with miR-NC inhibitor (150 nM) or pooled miR-17-92 inhibitors (25 nM each; 2-day time point). GAPDH was used as protein loading control. (F) Western blot analysis of BIM in lysates from AMO1 transduced with an empty lentiviral vector or an miR-17-92 lentiviral vector. GAPDH was used as protein loading control. (G) Western blot analysis of BIM (upper panel) and flow cytometric analysis of 7-AAD–stained cells (lower panel) in CRISPR/CAS9 genome-edited AMO1BIM− cells. GAPDH was used as protein loading control. Flow cytometry was performed after 6 days of exposure to MIR17PTi (1 µM) or scr-NC (1 µM). (H) Western blot analysis of BIM (upper panel) and flow cytometric analysis of 7-AAD–stained cells (lower panel) in CRISPR/CAS9 genome-edited U266MYC+/BIM− cells. GAPDH was used as protein loading control. Flow cytometry was performed after 6 days of exposure to MIR17PTi (2.5 µM) or scr-NC (2.5 µM). (I) Proposed model of MYC/miR-17-92 FFLs and MIR17PTi mechanism of action in MM cells. Data from 1 of 3 independent experiments are shown in each panel. *P < .05. NT, not targeting; PTR, posttranscriptional regulation; TR, transcriptional regulation.

Molecular mechanism underlying MIR17PTi proapoptotic activity. (A) 7-AAD flow cytometric assay of U266MYC−/U266MYC+ after 6 days of treatment with MIR17PTi (2.5 µM) or scr-NC (2.5 µM); western blotting of MYC protein in U266MYC− and U266MYC+ is reported. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as protein loading control (left). 7-AAD flow cytometric assay of P493-6 after 6 days of treatment with MIR17PTi (2.5 µM) or scr-NC (2.5 µM), in presence or absence of doxycycline (dox); western blotting of MYC protein in P493-6 cultured for 2 days with or without doxycycline is reported. GAPDH was used as protein loading control (middle). Trypan blue exclusion staining in MYC-ER human mammary epithelial cells (HMECs) 2 days after transfection with MIR17PTi (50 nM) or scr-NC (50 nM) and cultured with or without tamoxifen (tam) (right). (B) Western blotting of BIM in lysates from healthy donor peripheral blood mononuclear cells (PBMCs; n = 2), Jeko-1 (mantle cell lymphoma; BIM null), Daudi, Raji (Burkitt lymphoma), and indicated HMCLs. GAPDH was used as protein loading control. (C) Western blot analysis of BIM in lysates from pMM cells (patients 12 and 13; extramedullary MM) exposed to MIR17PTi for 6 days at indicated concentrations. GAPDH was used as protein loading control. (D) Western blotting of BIM in AMO1, NCI-H929, or INA-6 exposed for 6 days to MIR17PTi (AMO1, 1 µM; NCI-H929 and INA-6, 2.5 µM) or equimolar scr-NC. GAPDH was used as protein loading control. (E) Western blot analysis of BIM in lysates from AMO1 transfected with miR-NC inhibitor (150 nM) or pooled miR-17-92 inhibitors (25 nM each; 2-day time point). GAPDH was used as protein loading control. (F) Western blot analysis of BIM in lysates from AMO1 transduced with an empty lentiviral vector or an miR-17-92 lentiviral vector. GAPDH was used as protein loading control. (G) Western blot analysis of BIM (upper panel) and flow cytometric analysis of 7-AAD–stained cells (lower panel) in CRISPR/CAS9 genome-edited AMO1BIM− cells. GAPDH was used as protein loading control. Flow cytometry was performed after 6 days of exposure to MIR17PTi (1 µM) or scr-NC (1 µM). (H) Western blot analysis of BIM (upper panel) and flow cytometric analysis of 7-AAD–stained cells (lower panel) in CRISPR/CAS9 genome-edited U266MYC+/BIM− cells. GAPDH was used as protein loading control. Flow cytometry was performed after 6 days of exposure to MIR17PTi (2.5 µM) or scr-NC (2.5 µM). (I) Proposed model of MYC/miR-17-92 FFLs and MIR17PTi mechanism of action in MM cells. Data from 1 of 3 independent experiments are shown in each panel. *P < .05. NT, not targeting; PTR, posttranscriptional regulation; TR, transcriptional regulation.

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