Journal:

Article Title: The Cyclin A1-CDK2 Complex Regulates DNA Double-Strand Break Repair

doi: 10.1128/MCB.24.20.8917-8928.2004

Figure Lengend Snippet: Triple hybrid screening: interaction of cyclin A1 with Ku70/Ku80. (A) A yeast triple hybrid screen was performed with a Gal4-binding domain-cyclin A1 fusion protein as the bait, while human CDK2 was conditionally expressed. Screening was performed with a human testis cDNA library. (B) Two of the identified interacting clones contained a fragment of the human Ku70 cDNA. (C) Interaction of cyclin A1 and Ku70 in vitro. Baculovirus-expressed cyclin A1 or cyclin A1/CDK2 was incubated with GST, GST-Ku70, or GST-Ku80. After GST pulldown, binding was detected with anti-cyclin A1 antibody or anti-CDK2 antibody. (D) Deletion mutants of Ku70 were tested for their interaction with cyclin A1 in GST pulldown assays. Cyclin A1 binding was detected with anti-cyclin A1 Western blotting. The C terminus of Ku70 was required for the interaction with cyclin A1. (E) Interaction of cyclin A1 and Ku70 in vivo. Immunoprecipitations were performed with either anti-cyclin A1 or control immunoglobulin G. Ku70 coimmunoprecipitated with cyclin A1 in U937 cell lysate. (F) GST-Ku70, GST, or GST-Ku80 was incubated with baculovirus-expressed cyclin A1, CDK2, or cyclin A1/CDK2 in the presence of [γ-32P]ATP. Lysate from wild-type baculovirus-infected insect cells was used as a control. Following SDS-PAGE, the gel was autoradiographed. GST-Ku70 protein was clearly phosphorylated by cyclin A1/CDK2, whereas GST-Ku80 was not. The nonspecific higher band appeared in all three GST preparations (GST, GST-Ku70, and GST-Ku80).

Article Snippet: U937 cells were lysed in radioimmunoprecipitation (RIPA) buffer; 300 μg of cell lysate was used in the immunoprecipitation assays with 3 μg of primary antibody in 500 μl of RIPA buffer for 2 h at 4°C; 50 μl of a 50% slurry of protein A/G-Plus agarose (Santa Cruz Biotechnology) was added for another 1 h, and beads were washed three times with RIPA buffer.

Techniques: Binding Assay, cDNA Library Assay, Clone Assay, In Vitro, Incubation, Western Blot, In Vivo, Infection, SDS Page

Journal: PLoS Pathogens

Article Title: Mobilization of HIV Spread by Diaphanous 2 Dependent Filopodia in Infected Dendritic Cells

doi: 10.1371/journal.ppat.1002762

Figure Lengend Snippet: ( A ) Detection strategy of HIV-T. The HIV Gag polyprotein is presented in the context of the HIV open reading frames. The biarsenical fluorescent dye FlAsH is shown and binds to a 12 amino acid motif (in bold) at the C-Terminus of Matrix. The protease cleavage site between HIV Matrix and Capsid is highlighted by black scissors. ( B ) Detection strategy using HIV-iGFP. HIV-iGFP constructs encode eGFP at the C-terminus of HIV matrix and are flanked by 5′ and 3′ HIV protease cleavage sites (highlighted by black scissors). For generation of cell-free virus with comparable infectivity to WT HIV Gag and Gag-Pol are expressed in trans to the HIV iGFP genome (see bottom of panel; HIV Gag only shown) to increase viral infectivity in one round. ( C ) & ( D ) Rescuing infectivity of HIV-iGFP. ( C ) HIV iGFP was prepared by co-transfecting WT HIV or WT Gag and Gag -POL (psPAX2) with HIV iGFP at an equimolar ratio into the 293T cell line. Three days post transfection, supernatants were harvested, diluted 1/1000 and 200 ml was added to 1×10 3 TZM-bl cells (HeLa HIV indicator cell line), seeded 24 hours prior in a 96 well plate. % infectivity is relative to wild type HIV and calculated as the (co-transfections)/(HIV-WT alone)×100. Statistical differences are presented as p values. Standard deviations are derived from assays in triplicate. ( D ) Further titration of psPAX2∶HIV-iGFP. As in C. HIV-iGFP was co-transfected with psPAX2, but here at as a titration. Supernatants were subsequently titered using the TZM-bl cell line as in C. Standard deviations and p values also as per C. % infectivity relative to wild type HIV is calculated as in C. ( E ) DC were infected with an MOI of 0.1 with either WT HIV (left panel), WT HIV rescued HIV-iGFP virus (middle panel) or psPAX2 rescued HIV-iGFP (right panel) as outlined in . To determine total infection, infected DC were stained with anti-HIV-p24 antibody KC57-RD1. Gates in panels reflect eGFP signal from infected p24 high cells, with percentages from gates presented in the lower right corners. Note WT rescued HIV iGFP virus generates infected DC with diluted eGFP signal. ( F ) Infected DCs expressing HIV-iGFP 4 days post infection and co-cultured with autologous resting CD4 T cells at a ratio of 1 DC to 3 CD4 T cells (images are also representative for HIV-T). Filopodia are highlighted by dotted lines. Neighboring CD4 T cells that are in contact with filopodia are marked as (T) ( G ) HIV iGFP infected cells (HIV in white) have been fixed and stained for F-actin using phalloidin dye (red). Note all filopodia stained red, bear HIV at their terminal tips (All scale bars are 5 µm). ( H ) Average lengths of filopodia & VF across multiple DC donors. Infected or uninfected DCs (untreated U/T) were co-cultured with CD4 T cells as in F. Length of filopodia from the base at the plasma membrane to the tip was calculated in live infected and uninfected DC donors. VF and Filopodia lengths in infected and uninfected co-cultures from D1 & D2 are presented as a comparison. Filopodial lengths are representative of greater than n = 20 donors. VF and filopodia from infected and uninfected U937 cell line are also presented as a comparison.

Article Snippet: Knockdown at the protein level was verified by western blotting of U937 lysates using the Wasp mAb clone D1 (Santa Cruz) and goat-polyclonal sera (C-12) raised against the C-terminal of Diaph2 (Santa Cruz).

Techniques: Construct, Virus, Infection, Transfection, Derivative Assay, Titration, Staining, Expressing, Cell Culture, Clinical Proteomics, Membrane, Comparison

Journal: PLoS Pathogens

Article Title: Mobilization of HIV Spread by Diaphanous 2 Dependent Filopodia in Infected Dendritic Cells

doi: 10.1371/journal.ppat.1002762

Figure Lengend Snippet: DC were infected with HIV-iGFP and subsequent live imaging proceeded with infected DCs co-culture with CD4 T cells at a ratio of 1 DC to 3 CD4 T cells as outlined in . ( A ) Untethered VF engage in sweeping Arc trajectories. To illustrate the overall movement of VF, 8 frames of the Supplementary were superimposed. To highlight filopodia, a dashed line shadows the filopodial connection on VF as in . ( B ) 10 representative velocities of VF tips over time (lower graph) and corresponding movements from their point of origin (PO) (upper graph) ( C ) 10 representative velocities of filopodia from uninfected DC from the same donor. ( D ) Average velocities across entire VF trajectories across multiple DC donors and filopodial trajectories from untreated (U/T) controls (each point is the average VF Velocity over an entire 20 second trajectory). VF and filopodia from infected and untreated (U/T) U937 monocyte cell line is presented herein as a comparison. ( E ) Change in VF trajectories from Arc to Scan movements, when contacting CD4 T cells. The distance of the VF tip to the target T cells was calculated over time for 10 representative VF. Vertical red lines highlight the average scanning time of filopodia on the CD4 T cell membrane. Although scanning by normal filopdoia occurs, we could not resolve definitive trajectories as we did not have a tip marker equivalent to HIV on VF. ( F ) To illustrate the appearance of Scan trajectories in close contact with CD4 T cells, 3 frames have been taken from a live imaging time lapse experiment in , and single particle tracking over time highlighted in each frame. All scale bars are at 5 µm. All data is representative of in excess of 12 independent donors.

Article Snippet: Knockdown at the protein level was verified by western blotting of U937 lysates using the Wasp mAb clone D1 (Santa Cruz) and goat-polyclonal sera (C-12) raised against the C-terminal of Diaph2 (Santa Cruz).

Techniques: Infection, Imaging, Co-Culture Assay, Comparison, Membrane, Marker, Single-particle Tracking

Journal: PLoS Pathogens

Article Title: Mobilization of HIV Spread by Diaphanous 2 Dependent Filopodia in Infected Dendritic Cells

doi: 10.1371/journal.ppat.1002762

Figure Lengend Snippet: ( A ) To further delineate how VF pathway are formed, Wasp and Diaph2 was knockdown in the U937 cell line using shRNA. After 2 weeks of puromycin selection, resistant U937 cell lines were infected with HIV iGFP and VF lengths and trajectory velocities enumerated as in . P values are included to highlight significant differences in each variable. Protein knock-down for Wasp and Diaph2 are presented in . The house-keeping protein Gapdh is present below to normalize lysate loading. Data is representative of 4 independent infections using HIV iGFP. ( B ) To rule out manipulation of the Arp2/3 filopodial pathway, the U937 cell line was infected with HIV iGFP and two days post infection, infected cells were treated with the Abl/Src kinase inhibitor Dasatinib at 10 µM for 4 hours. Note, under these conditions Vaccinia actin tails do not form (data not shown). VF were then enumerated for lengths and trajectory velocities as outlined . ( C ) Accumulative single particle tracking for 1 minute of VF trajectories in scrambled controls (upper panel) versus Diaph2 knockdowns (lower panel). Note the confined trajectories in the absence of Diaph2. Diaph2 knockdown particle tracking is derived from . Scale bars are 5 µm. Data from knockdown experiments is representative of 4 independent HIV iGFP infections. ( D ) Fixed cell images of control shRNA (upper panel) and Diaph2 (lower panel) transduced cells infected with HIV iGFP. Note the significantly shorter VF lengths in Diaph2 knockdown U937 cells. Scale bars are 5 µm. Images are representative of 4 independent infections with HIV iGFP. ( E ) Attenuation of cell-cell transfer in Diaph2 knockdown U937. U937 were infected with HIV and 2 days post infection were stained for HIV p24 and enumerated by flow cytometry. After infections were verified to be equivalent, infected U937 cells were co-cultured at a ratio of 1∶5 with the T cell HIV indicator cell line JLTR-R5. Four days post infection, fluorescent images were acquired for the entire well and enumerated using Image J. Standard deviations represent co-cultures in triplicate. Data is representative of 3 independent infections. ( F ) VF form in the absence of HIV envelope. DCs were infected with either VSVg pseudotyped HIV iGFP or HIV iGFP -ENV-ve as outlined in . VF were then enumerated for lengths and trajectory velocities as outlined B. Data is representative of four independent infections. P values are presented for significant differences. ( G ) Deletion of HIV Nef leads to significantly lower VF frequency on DC. Enumeration of VF numbers over time in uninfected DCs (U/T), or HIV infected DCS with HIV − iGFP or HIV -NEF -iGFP. Each point represents live imaging of a VF bearing DC over a period of 2 minutes under imaging conditions outlined in . Accumulative data presented is equally drawn from 5 independent donors. Statistical significance is indicated by p values.

Article Snippet: Knockdown at the protein level was verified by western blotting of U937 lysates using the Wasp mAb clone D1 (Santa Cruz) and goat-polyclonal sera (C-12) raised against the C-terminal of Diaph2 (Santa Cruz).

Techniques: Knockdown, shRNA, Selection, Infection, Single-particle Tracking, Derivative Assay, Control, Staining, Flow Cytometry, Cell Culture, Imaging

Journal: Science Advances

Article Title: Targeting oncoproteins with a positive selection assay for protein degraders

doi: 10.1126/sciadv.abd6263

Figure Lengend Snippet: ( A ) Immunoblot and ( B ) RT-qPCR analysis of the NCI-H1876 SCLC cell line that endogenously expresses ASCL1 infected to express the indicated sgRNAs. n = 3 biological replicates. ( C and E ) Immunoblot and ( D and F ) RT-qPCR analysis of NCI-H1876 human SCLC cells (C and D) and 97-2 mouse SCLC cells (E and F) after treatment with the CDK2 PROTAC degraders (TMX-2138 and TMX-2172) or the indicated negative controls, all used at 500 nM for either 36 hours (C and D) or 8 hours (E and F). Neg Deg, negative control degrader ZXH-7035. n = 3 biological replicates. ( G ) Immunoblot analysis and ( H ) quantification of ASCL1 protein levels in 97-2 cells first treated with the CDK2 PROTAC degrader or negative control (500 nM) for 4 hours and then treated with cycloheximide (CHX) (150 μg/ml) for the indicated times. S.E., short exposure; L.E., long exposure. n = 4 biological replicates. In all experiments, error bars represent SD except in (H), where error bars represent SEM. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following compounds were purchased: POM (Selleck, #S1567), LEN (Selleck, #S1029), MG132 ( N -carbobenzyloxy- l -leucyl- l -leucyl- l -leucinal; Thermo Fisher Scientific, #47479020MG), MLN4924 (Active Biochem, #A-1139), MLN7243 (Thermo Fisher Scientific, #NC1129906), Spautin-1 (BioTechne; #5197/10), cycloheximide (VWR, #97064-724), BVdU (Chem-Impex International Inc., catalog no. 27735), actinomycin D (Thermo Fisher Scientific, #11805017), and dinaciclib (Selleck, #S2768).

Techniques: Western Blot, Quantitative RT-PCR, Infection, Negative Control