Journal: PLoS ONE

Article Title: Cyclophilin B Interacts with Sodium-Potassium ATPase and Is Required for Pump Activity in Proximal Tubule Cells of the Kidney

doi: 10.1371/journal.pone.0013930

Figure Lengend Snippet: A: Yeast two hybrid assay. CDNA for CypB was cloned in frame into the yeast expression vector (pGBKT7) that harbours the GAL4 activation domain. The recombinant plasmids plus Human Kidney Matchmaker cDNA Library were co-transformed into AH109 yeast strain. The co-transformed cells were selected on Ade-/Leu-/His-/Trp-, 10 µM Aminotriazol dropout plates to monitor for growth. The positive control represents co-transformation of p53 and T-antigen in two-hybrid expression vectors, pGBKT7-P53 and pGADT7 (BD Biosciences, Clontech), respectively. B: GST-pull-down assays. CypB was fused in frame with the GST gene. GST and GST-HCypB products were immobilized on Sepharose 4B and incubated with COS-7 cells lysates expressing Na/K-β1 (pCMV-HA-Na/K). The bound proteins were analyzed by immunoblotting with anti-Na/K-β1 rabbit polyclonal antibody. C–E: Co-Immunoprecipitation assays. HK-2 lysates were immunoprecipitated with mouse monoclonal antibodies against Na/K-β1 or Na/K-α1 and rabbit polyclonal antibody against CypB. The immunoprecipitates were subjected to Western blotting analyses, as indicated in the figure. As control, ChromePure mouse IgG and ChromePure rabbit IgG were used. Figures 1B to 1E are representative of at least three independent experiments.

Article Snippet: BD Biosciences, clontech, Palo Alto, CA), was subcloned into the BglII restriction endonuclease site of pCMV-HA mammalian expression vector (BD Biosciences, clontech, Palo Alto, CA) to get the pCMV- Na/K-β1 -HA construct.

Techniques: Y2H Assay, Clone Assay, Expressing, Plasmid Preparation, Activation Assay, Recombinant, cDNA Library Assay, Transformation Assay, Positive Control, Incubation, Western Blot, Immunoprecipitation

Journal:

Article Title: BLIMP1 is a tumor suppressor gene frequently disrupted in activated B cell like diffuse large B cell lymphoma

doi: 10.1016/j.ccr.2010.10.030

Figure Lengend Snippet: (A) Distribution of truncating mutations along the BLIMP1 protein, with known functional domains annotated. Aci=acidic domain; PR=PR domain; Pro-rich=proline-rich domain (see also Table S1). (B) Percentage of cases with truncating BLIMP1 mutations in various DLBCL subtypes classified by gene expression profiling (GEP) or by immunohistochemistry (IHC)(see text). (C) dChip SNP inferred copy number heatmap of the 6q15-q22.1 region in ABC-DLBCL cases and three normal DNA controls. Cases harboring homozygous deletions or truncating mutations are denoted by symbols (see also Figure S1). The region indicated by an asterisk is shown at higher magnification in panel (D) for the three homozygously deleted cases (and two normal DNAs), with the approximate position of the BLIMP1, ATG5 and PREP genes on the left.

Article Snippet: For luciferase reporter assays, 293T cells were co-transfected with 10 ng of wild-type or mutant pCMV-HA- BLIMP1 vectors, 10 ng of CIITA-luc reporter construct and 0.1 ng of the TK-RL Renilla reporter as control for transfection efficiency (Promega, Madison, WI).

Techniques: Functional Assay, Expressing, Immunohistochemistry

Journal:

Article Title: BLIMP1 is a tumor suppressor gene frequently disrupted in activated B cell like diffuse large B cell lymphoma

doi: 10.1016/j.ccr.2010.10.030

Figure Lengend Snippet: (A) Distribution of missense mutations along the BLIMP1 protein; in red, mutants that showed an effect in any of the assays performed in (B–D)(see also Table S2). (B) Western blot (top) and semi-quantitative RT-PCR (bottom) analysis of exogenous BLIMP1 expression in 293T cells transfected with equimolar amounts of vectors expressing HA-tagged wild-type or mutant BLIMP1 alleles. (C) Analysis of exogenous BLIMP1 protein expression in 293T cells transfected with the indicated mutant alleles and treated with cycloheximide for 2, 4 or 8 hours. Data were quantitated by densitometric analysis, normalized to β-actin levels, and graphed relative to time zero (top). The western blot analysis is shown on the bottom panel. (D) Trans-repression activity of wild-type and mutant BLIMP1 proteins in 293T cells co-transfected with a luciferase reporter construct driven by the human CIITA promoter (region −545 to +123, encompassing a consensus BLIMP1 binding site at position −180)(top). Luciferase activities are represented as percent change relative to the basal activity of the reporter (set to 100), after normalization to Renilla luciferase activity (mean ± SD, as obtained from three independent experiments). In the bottom panel, western blot analysis using anti-HA antibodies monitors for the corresponding exogenous BLIMP1 expression levels; note that, for the three unstable mutants, higher amounts of plasmid DNA were transfected to achieve comparable levels. Nevertheless, expression of the P48R mutant protein remained significantly lower than wild-type, presumably due to its marked instability (see also Figure S2).

Article Snippet: For luciferase reporter assays, 293T cells were co-transfected with 10 ng of wild-type or mutant pCMV-HA- BLIMP1 vectors, 10 ng of CIITA-luc reporter construct and 0.1 ng of the TK-RL Renilla reporter as control for transfection efficiency (Promega, Madison, WI).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Transfection, Mutagenesis, Activity Assay, Luciferase, Construct, Binding Assay, Plasmid Preparation

Journal:

Article Title: BLIMP1 is a tumor suppressor gene frequently disrupted in activated B cell like diffuse large B cell lymphoma

doi: 10.1016/j.ccr.2010.10.030

Figure Lengend Snippet: (A) Proliferative capacity of BJAB B cells transduced with lentiviral vectors expressing the indicated BLIMP1 proteins along with GFP, as assessed by an MTT assay on sorted GFP+ cells. Shown are representative data from one of two independent experiments performed in duplicate (mean ± SD) (see also Figure S3). (B) Left, representative flow cytometric analysis of BJAB B cells transduced with the indicated vectors and stained for incorporated BrdU and 7-amino-actinomycin D (7-AAD). Region gates define cells residing in G0-G1, S and G2-M phases of the cell cycle. The G0-G1 population was quantitated relative to empty vector-transduced cells (set at 1), and the mean ± SD from two independent experiments is shown below. (C) Expression of the BLIMP1 targets ID3 and CIITA in sorted GFP+ BJAB B cells, as determined by quantitative real-time RT-PCR (n=3; mean ± SD). Levels were normalized to both BLIMP1 and GAPDH, and are shown as fold changes relative to vector-transduced cells (set as 1). (D) Representative flow cytometric analysis of CD138 and B220 staining in Blimp1CD19KO splenic B cells, reconstituted with the indicated vectors and stimulated to undergo plasma cell differentiation by LPS treatment for 3 days, as compared to wild-type (Blimp1CD19WT) B cells. The percentage of cells in the gated (plasma cell) population is shown. Data from two independent experiments are quantitated in the bottom panel (mean ± SD). Mutants that showed an effect in any of the assays (A-D) are indicated in red.

Article Snippet: For luciferase reporter assays, 293T cells were co-transfected with 10 ng of wild-type or mutant pCMV-HA- BLIMP1 vectors, 10 ng of CIITA-luc reporter construct and 0.1 ng of the TK-RL Renilla reporter as control for transfection efficiency (Promega, Madison, WI).

Techniques: Transduction, Expressing, MTT Assay, Staining, Plasmid Preparation, Quantitative RT-PCR, Cell Differentiation

Journal:

Article Title: BLIMP1 is a tumor suppressor gene frequently disrupted in activated B cell like diffuse large B cell lymphoma

doi: 10.1016/j.ccr.2010.10.030

Figure Lengend Snippet: (A) Immunofluoresence analysis of BLIMP1 (green), IRF4 (red) and CD20 (blue) expression in normal GC cells of a human tonsil (scale bar, 325μm; inset, 150μm). (B) IRF4 (brown) and BLIMP1 (blue) immunostaining in representative ABC-DLBCL cases displaying a normal expression pattern (IRF4+BLIMP1+)(left panels) or specific lack of BLIMP1 expression (IRF4+BLIMP1−)(right panels)(scale bar: 125 μm). The percentage of cases in each group is provided below. Six additional cases (12%) were negative for expression of both proteins (not shown; see also Table S1 and S2 for a detailed characterization of individual cases). (C) Distribution of BLIMP1 and BCL6 structural alterations in IRF4+ ABC-DLBCL. Columns represent individual patients, with color-codes indicating the presence or absence of the corresponding feature. (D) BLIMP1 mRNA (left) and protein (right) levels in the BCL6-translocated RCK8 cell line, transduced with lentiviral vectors expressing a control shRNA (shCtrl) or a BCL6-specific shRNA (shBCL6). BLIMP1 mRNA levels were determined by quantitative real-time RT-PCR and are shown as fold change relative to shCtrl-transduced cells, after normalization for GAPDH (n=3; mean ±SD). Western blot analysis of BCL6 controls for efficient BCL6 knockdown (see also Figure S4). (E) Overall frequency of BLIMP1 and BCL6 structural alterations in ABC-DLBCL. The unknown category denotes cases that lack BLIMP1 protein expression, in the absence of BLIMP1 or BCL6 structural alterations. Two cases carrying both BCL6 translocations and biallelic BLIMP1 inactivation were included into the “BLIMP1 mutation + deletion” category.

Article Snippet: For luciferase reporter assays, 293T cells were co-transfected with 10 ng of wild-type or mutant pCMV-HA- BLIMP1 vectors, 10 ng of CIITA-luc reporter construct and 0.1 ng of the TK-RL Renilla reporter as control for transfection efficiency (Promega, Madison, WI).

Techniques: Expressing, Immunostaining, Transduction, shRNA, Quantitative RT-PCR, Western Blot, Mutagenesis

Journal:

Article Title: BLIMP1 is a tumor suppressor gene frequently disrupted in activated B cell like diffuse large B cell lymphoma

doi: 10.1016/j.ccr.2010.10.030

Figure Lengend Snippet: Representative spleen and lymph node sections from Blimp1 knock-out mice presenting with a spectrum of lymphoproliferative disorders, including MZBCH, LPD, and overt DLBCL, as compared to a wild-type control (see results for a detailed histological description, and Figure S6 for additional data). Tissues were stained with hematoxylin and eosin (H&E) or immunostained with antibodies against the B220 pan-B cell marker, IRF4 and the NF-κB subunit p50, as indicated (Scale bar, 1250 μm; inset, 125 μm; scale is 50 μm for p50 stain).

Article Snippet: For luciferase reporter assays, 293T cells were co-transfected with 10 ng of wild-type or mutant pCMV-HA- BLIMP1 vectors, 10 ng of CIITA-luc reporter construct and 0.1 ng of the TK-RL Renilla reporter as control for transfection efficiency (Promega, Madison, WI).

Techniques: Knock-Out, Staining, Marker