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Blood, Vol. 113, Issue 26, 6669-6680, June 25, 2009
MicroRNAs 15a and 16 regulate tumor proliferation in multiple myeloma
Blood Roccaro et al.
113: 6669
Supplemental materials for: Roccaro et al
Files in this Data Supplement:
- Table S1. Main clinical characteristics of MM patients (PDF, 12.6 KB)
- Table S2. Predicted targets modulated by deregulated miRNA expression in MM patients (PDF, 18.1 KB)
- Figure S1 (JPG, 365 KB)
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(A) Efficiency of pre–miRNA-15a and –16-1 probe transfection in MM cells. MM.1S and RPMI8226 cell lines (pre–miRNA-15a, –16-1−, control probe-transfected cells) were harvested 48 and 72 hours after transfection. Not-transfected MM.1S and RPMI8226 cells were used as controls. Detection of miRNA-15a and -16 was evaluated by stem-loop qRT-PCR. (B) Nocodazole does not induce cytotoxicity on either pre–miRNA-15a−, –16-1, or control probe-transfected MM cells. MM.1S and RPMI8226 cell lines were cultured in presence or absence of nocodazole (80 nM) for 24 h, and cytotoxicity were assessed by MTT assay. (C) Efficiency of pre–miRNA-15a and –16-1 probe transfection in MM cells isolated from mice BM. Detection of miRNA-15a and –16-1 was evaluated from BM of mice injected with either pre–miRNA-15a, –16-1 probe−, or control probe-transfected MM.1S cells, by using stem-loop qRT-PCR for human miRNA-15a and -16.
- Figure S2. miRNA-15a and –16-1 target RPMI8226 cells (JPG, 531 KB)
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(A) MM cells (pre–miRNA-15a-, –16-1–precursors probe− , control probe-transfected, and non-transfected RPMI8226) were harvested at 24–48–72 hours after transfection; DNA synthesis and cytotoxicity were assessed by thymidine uptake and MTT assays, respectively. Non-transfected RPMI8226 cells were used as controls. P values are indicated. (B) Cells were first arrested and synchronized in G2/M phase by growth in 80nM nocodazole for 16 h. Cells were then washed and re-growth using fresh media. After 6 hours, cell cycle analysis was performed by propidium iodide staining. (C) MM cells (pre–miRNA-15a-, –16-1 precursors probe−, control probe-transfected RPMI8226) were harvested at 24 hours after transfection and treated with and without TNF-α (10 ng/mL) for 20 minutes; non-transfected RPMI8226 cells were used as control (ctrl). NF-kB-p65, p50, -p52, -RelB transcription factor-binding to its consensus sequence on the plate-bound oligonucleotide was measured in nuclear extracts. All results represent means (±sd) of triplicate experiments. (D) VEGF concentrations were measured in triplicate, by ELISA, in conditioned media obtained from MM cells (pre–miRNA-15a−,–16-1 precursors probe−, control probe-transfected RPMI8226).
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