![Figure 3. miRNA-155 modulates proliferation and cell cycle of WM cells. (A,B) WM cells (miRNA-155 knockdown probe–transfected, control probe–transfected BCWM.1 cells) were harvested at 24 and 48 hours after transfection; DNA synthesis and cytotoxicity were assessed by thymidine uptake and MTT assays, respectively. Nontransfected BCWM.1 cells were used as controls (ctrl). *P < .001; **P < .001. (C) Cells were first arrested and synchronized in G2/M phase by growth in 80 nM nocodazole for 16 hours. Cells were then washed and regrown using fresh media. After 6 hours, cell-cycle analysis was performed by propidium iodide staining. (D) Purified cRNA (15 μg) isolated from WM cells (control probe–transfected, miRNA-155 knockdown probe–transfected BCWM.1 cells) was hybridized to HG-U133Plus2.0 GeneChip (Affymetrix). (i) Up-regulation of cyclin-dependent kinase inhibitors and down-regulation of cyclins and cyclin-dependent kinases. (ii) Up-regulation of p53 and p53 family members and down-regulation of p53 negative regulators. Fold change is shown by the intensity of induction (red) or suppression (blue). (E) BCWM.1 cells (miRNA-155 knockdown probe transfected, control probe transfected) were harvested at 8 hours after transfection. Whole-cell lysates were subjected to Western blotting using anti–phospho (p)-ERK, –p-AKT, -AKT, –p-GSK3α/β, –p-S6R, and -actin antibodies; nontransfected BCWM.1 cells were used as controls (ctrl). (F) BCWM.1 cells (miRNA-155 knockdown probe transfected, control probe transfected) were harvested at 8 hours after transfection and treated with and without TNF-α (10 ng/mL) for 20 minutes; nontransfected BCWM.1 cells were used as control (ctrl). NF-κBp65 transcription factor binding to its consensus sequence on the plate-bound oligonucleotide was measured in nuclear extracts. Wild-type and mutant are wild-type and mutated consensus competitor oligonucleotides, respectively. All results represent means (±] SD) of triplicate experiments. (G) BCWM.1 cells (miRNA-155 knockdown probe transfected, control probe transfected) were harvested at 8 hours after transfection and treated with and without TNF-α (10 ng/mL) for 20 minutes; nontransfected BCWM.1 cells were used as control (ctrl). Immunocytochemical analysis was assessed using anti–p-NF-κBp65 antibody, with DAPI used to stain nuclei.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/113/18/10.1182_blood-2008-09-178228/6/zh80100931820003.jpeg?Expires=1724269695&Signature=j1B-MU4JmgNWuFrWBD5yDFCyBNMQwEqlUD2i~il9DZawoEAEuvXHHXlpfC6RWewglYlWWKayAI4WBa94cScQrpBhX7qZOwou4gOoxNYmtJ5rEj0lNA7q3AISSL7hwvaoRLywc7HzpuRkat0t68JCX9hReK8Gax7H9ZNuifVfiKWTg8~dy4u92AK8CwM9o2Tww4KZplCUJj6H~eAYXOvi0-K4xjtvVRp6zouo-XX9rNb6DNbzzl1VI9NpDL4LKVnA4Ih2j4PKIF2Iu9ipGT-xe5ZO-wYw9p-XzeoO9wMAG110G6W0jAwy2xn9lQoicObfVE-2kNTWByKDcixtxXE60Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
miRNA-155 modulates proliferation and cell cycle of WM cells. (A,B) WM cells (miRNA-155 knockdown probe–transfected, control probe–transfected BCWM.1 cells) were harvested at 24 and 48 hours after transfection; DNA synthesis and cytotoxicity were assessed by thymidine uptake and MTT assays, respectively. Nontransfected BCWM.1 cells were used as controls (ctrl). *P < .001; **P < .001. (C) Cells were first arrested and synchronized in G2/M phase by growth in 80 nM nocodazole for 16 hours. Cells were then washed and regrown using fresh media. After 6 hours, cell-cycle analysis was performed by propidium iodide staining. (D) Purified cRNA (15 μg) isolated from WM cells (control probe–transfected, miRNA-155 knockdown probe–transfected BCWM.1 cells) was hybridized to HG-U133Plus2.0 GeneChip (Affymetrix). (i) Up-regulation of cyclin-dependent kinase inhibitors and down-regulation of cyclins and cyclin-dependent kinases. (ii) Up-regulation of p53 and p53 family members and down-regulation of p53 negative regulators. Fold change is shown by the intensity of induction (red) or suppression (blue). (E) BCWM.1 cells (miRNA-155 knockdown probe transfected, control probe transfected) were harvested at 8 hours after transfection. Whole-cell lysates were subjected to Western blotting using anti–phospho (p)-ERK, –p-AKT, -AKT, –p-GSK3α/β, –p-S6R, and -actin antibodies; nontransfected BCWM.1 cells were used as controls (ctrl). (F) BCWM.1 cells (miRNA-155 knockdown probe transfected, control probe transfected) were harvested at 8 hours after transfection and treated with and without TNF-α (10 ng/mL) for 20 minutes; nontransfected BCWM.1 cells were used as control (ctrl). NF-κBp65 transcription factor binding to its consensus sequence on the plate-bound oligonucleotide was measured in nuclear extracts. Wild-type and mutant are wild-type and mutated consensus competitor oligonucleotides, respectively. All results represent means (±] SD) of triplicate experiments. (G) BCWM.1 cells (miRNA-155 knockdown probe transfected, control probe transfected) were harvested at 8 hours after transfection and treated with and without TNF-α (10 ng/mL) for 20 minutes; nontransfected BCWM.1 cells were used as control (ctrl). Immunocytochemical analysis was assessed using anti–p-NF-κBp65 antibody, with DAPI used to stain nuclei.
