Journal: Scientific Reports

Article Title: The interactome of a family of potential methyltransferases in HeLa cells

doi: 10.1038/s41598-019-43010-2

Figure Lengend Snippet: Experimental workflow. ( a ) ORFs of METTL proteins were cloned as GFP fusions under the control of a DOX-inducible promoter for targeted single copy integration into HeLa FRT cells. Whole cell extracts and/or nuclear extracts were prepared from GFP-METTL or GFP (control) expressing cells. GFP pull-downs were performed in triplicate followed by mass spectrometry analysis. Analysis of raw data was performed in MaxQuant (version 1.5.1.0) and Andromeda. Data filtering and generation of volcano plots was done essentially as described using a one-way ANOVA test with log2 fold change (FC) > 8 and false discovery rate (FDR) < 0.05 as thresholds. ( b ) Volcano plot of GFP-MBD3 interacting proteins as an example of successful complex identification using this protocol. Significant interactors are indicated. Volcano plots of ( c ) METTL3 and ( d ) METTL14 interactors. The log2 FC of GFP fusions to control in label-free quantification are plotted against the −log10 of the FDR calculated by a permutation-based FDR adapted t-test. The baits are indicated in red.

Article Snippet: GFP, GFP-METTL8 and GFP-METTL16 were purified from stable HeLa FRT cell lines using GFP nanobody sepharose beads (Chromotek).

Techniques: Clone Assay, Control, Expressing, Mass Spectrometry, Quantitative Proteomics

Journal: Scientific Reports

Article Title: The interactome of a family of potential methyltransferases in HeLa cells

doi: 10.1038/s41598-019-43010-2

Figure Lengend Snippet: Confirmation of METTL9 interactor and enzymatic activity of GFP-METTLs. ( a , b ) Validation of interaction between METTL9 and CANX by co-IP. ( a ) CANX is detected in GFP-METTL9 IP (a, lane 5) but not GFP IP (a, lane 2). 10 μl of GFP trap and 2 mg of whole cell extract were used. 20 μg of Input material were loaded for a comparison. ( b ) GFP-METTL9 (left panel, line 2) but not GFP (right panel, line 3) can be detected by immuno-blot with GFP antibody in the CANX IP. 10 μg of calnexin antibody and 4 mg of whole cell extract were used. 200 μg of Input material were loaded for comparison. No antibody (beads alone) used as control. ( c ) In vitro methyltransferase assays demonstrating that our GFP-METTL8 and GFP-METTL16 purifications have the expected RNA methyltransferase activity. GFP (as a control) and GFP-fusion proteins were purified from corresponding DOX-induced HeLa FRT cell lines and used in an in vitro methyltransferase assay on total RNA from HeLa cells as a substrate and 3H-SAM as a methyl-donor. After purification of the RNA, counts per minute (CPM) were quantified by liquid scintillation counting. Ratio of CPM measured for reactions with GFP-METTL fusion proteins relative to GFP control are plotted. Data are shown as mean ± SD from three replicates.

Article Snippet: GFP, GFP-METTL8 and GFP-METTL16 were purified from stable HeLa FRT cell lines using GFP nanobody sepharose beads (Chromotek).

Techniques: Activity Assay, Biomarker Discovery, Co-Immunoprecipitation Assay, Comparison, Control, In Vitro, Purification

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: The HIV permissive HeLa cell TZMbl (Courtesy of Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc, was then infected at an MOI of 1 and 7 days post transduction, high Lifeact mCherry single cells clones were sorted into 96 well plates using a FACSAria (Becton Dickinson, San Jose, CA).

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