miR-155 regulates HGAL expression and increases lymphoma cell motility by Liat Nadav Dagan, Xiaoyu Jiang, Shruti Bhatt, Elena Cubedo, Klaus Rajewsky, and Izidore S. Lossos Blood Volume 119(2):513-520 January 12, 2012 ©2012 by American Society of Hematology

miR-155 down-regulates HGAL expression by binding to specific sites in HGAL 3′-UTR. miR-155 down-regulates HGAL expression by binding to specific sites in HGAL 3′-UTR. (A) Effect of hsa-miR-155 overexpression on native HGAL protein levels in Raji, MC116, and VAL cell lines at 48 hours after transfection, assessed by Western blot. Actin levels were used as loading control in all cases. (B-C) Effect of hsa-miR-155 overexpression on the mRNA levels of HGAL in Raji (B) and MCL116 (C) cell lines measured by real-time PCR using TaqMan Gene Expression Assays (Applied Biosystems) at 24, 48, and 72 hours after transfection. Values of triplicate wells are represented as fold expression with respect to the nontargeting control transfection. Overexpression of hsa-miR-155 was confirmed by TaqMan MicroRNA Assays (C right panel and D right panel), expressed as fold increase over the control transfection. Error bars represent SEM. (D) Dual luciferase activity of reporter plasmids with the wild-type or mutated 3′-UTR of HGAL fused to the luciferase gene following hsa-miR-155 cotransfection in HeLa cells. The black columns represent cotransfections with hsa-miR-155; and the gray columns, the cotransfection of the same reporter vector with the nontargeting control. Mutations of the putative binding sites are represented as M1 and M2 3′-UTR. Values are normalized to the value of each control, which is noted as 100%. *Significant difference (P < .05). Error bars represent SEM. (A-D) Results are representative of 3 independent experiments. Liat Nadav Dagan et al. Blood 2012;119:513-520 ©2012 by American Society of Hematology

miR-155 increases lymphoma cell motility. miR-155 increases lymphoma cell motility. (A) Raji, VAL, and MC116 cells were transfected with hsa-miR-155 or nontargeted control. Forty-eight hours later, the cells were used for SDF-1 chemotaxis assay performed in triplicate, as described in “Chemotaxis assays.” Data are mean ± SE. *Significant difference (P < .05). Western blot confirms down-regulation of HGAL protein levels in the hsa-miR-155–transfected cells. (B) Representative pictures demonstrating spontaneous movement paths of Raji cells, transfected with hsa-miR-155 or with nontargeted control. Forty-eight hours after transfection, cells were seeded on fibronectin-coated 6-well plates. Time lapse images were acquired every 2 minutes for 1 hour. Top portion: Motility tracks (in green) of selected cells (in red). Bottom portion: Motility tracks of 50 randomly selected cells over 1 hour, measured, and graphed using the Track Points. Each track was assigned a different random color. Timelapse images were acquired with a BD Pathway 855 HCS Bioimager (BD Biosciences) with an Olympus 10×/0.3NA objective lens and ORCA-ER CCD camera. Images were saved as 1344 × 1024 pixel TIFF images, opened in MetaMorph 5.07 (Universal Imaging–Molecular Devices) and generated the video and measured and graphed using Track Points. Data was exported to Microsoft Excel 2007 by dynamic data exchange. (C) Average velocity of 50 cells in each treatment group. *Significant difference (P < .05). Error bars represent SEM. Liat Nadav Dagan et al. Blood 2012;119:513-520 ©2012 by American Society of Hematology

miR-155 affects RhoA activation and F-actin polymerization. miR-155 affects RhoA activation and F-actin polymerization. (A) Raji, VAL, and SUDHL6 lymphoma cells, transfected with hsa-miR-155 or control nontargeting miRNA 48 hours before the experiment, were starved for 8 hours and then treated with LPA (1.5 μg/mL) for 45 seconds. Cellular lysates were prepared, and RhoA pull-down assay was performed. HGAL knockdown and equal loading were confirmed by Western blot of HGAL, RhoA, and actin antibodies. Densitometry analysis of normalized RhoA-GTP to total RhoA is presented. The values in the control samples were arbitrarily defined as 1. Results are representative of 3 independent experiments. (B) Assay for polymerized F-actin. Raji, VAL, and SUDHL6 lymphoma cells, transfected with hsa-miR-155 or nontargeted control miRNA 48 hours before the experiment, were starved for 8 hours and then left unstimulated or treated with LPA (1.5 μg/mL) for 45 seconds followed by staining with Alexa-488 phalloidin and analyzed by flow cytometry. Liat Nadav Dagan et al. Blood 2012;119:513-520 ©2012 by American Society of Hematology

Chemotaxis of bic/miR-155−/− mice splenic B cells. Chemotaxis of bic/miR-155−/− mice splenic B cells. B-cell splenocytes form bic/miR-155−/− or control mice were used for SDF-1 and IL-6 chemotaxis assay performed in triplicate, as described in “Chemotaxis assays.” Liat Nadav Dagan et al. Blood 2012;119:513-520 ©2012 by American Society of Hematology

miR-155 regulates expression of RTKN2. miR-155 regulates expression of RTKN2. (A) Effect of hsa-miR-155 overexpression on native RTKN2 and myosin light chain kinase protein levels in Raji, VAL, and MC116 cell line at 48 hours after transfection, assessed by Western blot. Actin levels were used as loading control. Densitometry analysis of normalized RTKN2 and myosin light chain kinase protein to actin are presented. The values in the control samples were arbitrarily defined as 1. Results are representative of 3 independent experiments. (B) Dual luciferase activity of reporter plasmids with the wild-type or mutated 3′-UTR of RTKN2 fused to the luciferase gene following hsa–miR-155 cotransfection in HeLa cells. The black columns represent cotransfections with hsa–miR-155; and the gray columns, the cotransfection of the same reporter vector with the nontargeting control. Mutations of the putative binding sites are represented as M1 and M2 3′-UTR and concomitant mutation of both sites as M1 + 2 3′-UTR. Values are normalized to the value of each control, which is noted as 100%. *Significant difference (P < .05). Error bars represent SEM. Liat Nadav Dagan et al. Blood 2012;119:513-520 ©2012 by American Society of Hematology

HGAL mediates miR-155 effects on human lymphoma cell motility. HGAL mediates miR-155 effects on human lymphoma cell motility. VAL lymphoma cells were transfected with control nontargeting miRNA and hsa-miR-155 alone or with pcDNA3.1-HGAL plasmid. Forty-eight hours later, the cells were used for SDF-1 chemotaxis assay performed in triplicate, as described in “Chemotaxis assays.” Data are mean ± SEM of triplicates. *Significant difference (P < .05). Western blot confirms down-regulation of endogenous HGAL protein levels in hsa-miR-155–transfected cells and rescue of HGAL expression in cells transfected with pcDNA3.1-HGAL plasmid. Densitometry analysis of normalized endogenous HGAL to actin is presented. The values in the control samples were arbitrarily defined as 1. Results are representative of 2 independent experiments. Liat Nadav Dagan et al. Blood 2012;119:513-520 ©2012 by American Society of Hematology