Journal: Developmental cell

Article Title: Single-Cell and Population-Level Analyses Using Real-Time Kinetic Labeling Couples Proliferation and Cell Death Mechanisms

doi: 10.1016/j.devcel.2019.08.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, Peptides, and Recombinant Proteins ABT-737 Selleck Chemicals Cat#: S1002 mTNFα Peprotech Cat#: 315-01A mTRAIL Peprotech Cat#: 315-19 STYO21 Thermo Fisher Scientific Cat#: S7556 YOYO-3 Iodide Thermo Fisher Scientific Cat#: Y3606 zVAD-fmk APExBIO Cat#: A1902 Experimental Models: Cell Lines Bax +/+ Bak +/+ SV40 transformed MEF ATCC Cat#: CRL-2907; RRID: CVCL_U630 Bax −/− Bak −/− SV40 transformed MEF ATCC Cat#: CRL-2913; RRID: CVCL_U626 Cyld −/− SV40 transformed MEF Adrian Ting (Icahn School of Medicine at Mount Sinai, NY, USA) O’Donnell et al., 2011 Mlkl −/− adult ear fibroblasts Warren Alexander (University of Melbourne, Melbourne, Australia) Murphy et al., 2013 Recombinant DNA pProEx.Htb.annexinV Seamus Martin (Trinity College, Dublin, Ireland) Accession#: {"type":"entrez-nucleotide","attrs":{"text":"NM_001154","term_id":"1519243621","term_text":"NM_001154"}} NM_001154 ; Logue et al., 2009 Software and Algorithms Excel v16.16.9 Microsoft N/A ImageJ/Fiji v2.0.0-rc-69/1.52n National Institutes of Health N/A IncuCyte ZOOM v2018A Essen Biosciences N/A Prism v8.1.1 Graphpad Software N/A Sequential labeling pipeline This paper https://doi.org/10.5281/zenodo.3458574 Open in a separate window KEY RESOURCES TABLE SPARKL workflows use live-cell imagers to capture the kinetics of cell death in real time Multi-parametric analyses of cell death kinetics reveal mechanisms for comparative study Multiplex workflows accounts for cell-inherent or drug-induced proliferation changes Single-cell analyses quantify differential response to drugs in isogenic cell populations

Techniques: Recombinant, Transformation Assay, Software, Labeling

Journal: Nucleic Acids Research

Article Title: CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis

doi: 10.1093/nar/gkac114

Figure Lengend Snippet: CFIm KD increases the activity of miRNAs. ( A ) Genome browser tracks showing the coverage of DICER1 TE (bottom track) by RNA-seq reads from two replicate experiments for each condition, with the two PAS that were quantified for this gene marked by black lines. The conditions are color-coded (as in Figure ) and also indicated on the y-axis. Y-axis shows the smoothened number of reads mapping along the TE, calculated by the GViz R package. ( B ) RNA fluorescence in situ imaging of DICER1 isoforms in Control and CFIm25 KD HEK293 cells with probes corresponding to the common region of the long and short 3′ UTRs (red) or to the region between the proximal and distal cleavage sites, thus present exclusively in the long 3′ UTR (green). Nuclei are marked with DAPI. Zoom-ins of the regions marked with dashed boxes are further shown both with the individual and merged channels. A snapshot of a digital representation of the actual image as processed in IMARIS is also depicted for reference. ( C ) Quantification of the copy number of the long and short 3′ UTR isoforms of DICER1 in the nucleus (left plot) and cytoplasm (right plot) of Control, CFIm25 and CFIm68 KD cells. Colocalization of the red and green signals reveals the presence of the long 3′ UTR isoform (yellow) whereas the signal from the red probe only reveals the presence of the shorter 3′ UTR isoform. mRNA copy numbers were estimated separately from the nucleus (overlapping with DAPI) and cytosol. Segregation of the signal was performed with IMARIS (see Methods). ( D ) Representative western blot showing the DICER1 expression in the Control, CFIm25 and CFIm68 KD cells. The quantification is relative to GAPDH. ( E ) qPCR measurements of let-7, miR-92a, miR-16 and miR-19b expression in CFIm25/68 KD cells relative to Control. ΔΔct values were calculated relative to U6 snRNA and then relative to the Control cells (where the ratio was set to 1). ( F ) Normalized Renilla luciferase expression of reporter mRNAs carrying binding sites for miR-16 and miR-92a in their 3′ UTRs, in Control, CFIm25 and CFIm68 KD cells, respectively. The Firefly luciferase expressed from the same construct was used as normalization control.

Article Snippet: Detection and analysis of spots were performed using automated pipelines developed in image analysis software IMARIS (BITPLANE).

Techniques: Activity Assay, RNA Sequencing, Fluorescence, In Situ, Imaging, Control, Western Blot, Expressing, Luciferase, Binding Assay, Construct

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

doi: 10.1073/pnas.2114994119

Figure Lengend Snippet: Model of MT growth informed by cryo-EM and TIRF microscopy observations.

Article Snippet: We optimized our cryo-EM image analysis pipeline to deal with some unexpected challenges ( SI Appendix , Fig. S2 ; see Results ).

Techniques: Cryo-EM Sample Prep, Microscopy