lifeact mcherry sequence  (Addgene inc)

Journal: eLife

Article Title: SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance

doi: 10.7554/eLife.64370

Figure Lengend Snippet: Zebrafish MCR:EGFP (unpigmented) and MCR:SATB2 (pigmented) melanoma phenotype.

Article Snippet: Lentiviral particles were produced by co-transfection of 293 T cells with sequence verified pINDUCER20-SATB2, pLentiCMV_SATB2_Blast, or pTK93_Lifeact (Addgene plasmid #46357 was a gift from Iain Cheeseman), and packaging plasmids pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260, both gifts from Didier Trono), using FuGENE HD (Promega).

Techniques:

Journal: eLife

Article Title: SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance

doi: 10.7554/eLife.64370

Figure Lengend Snippet: Time-lapse video recording of Oregon Green 488-labeled gelatin degradation upon SATB2 induction in human melanoma cell line A375.

Article Snippet: Lentiviral particles were produced by co-transfection of 293 T cells with sequence verified pINDUCER20-SATB2, pLentiCMV_SATB2_Blast, or pTK93_Lifeact (Addgene plasmid #46357 was a gift from Iain Cheeseman), and packaging plasmids pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260, both gifts from Didier Trono), using FuGENE HD (Promega).

Techniques:

Journal: eLife

Article Title: SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance

doi: 10.7554/eLife.64370

Figure Lengend Snippet: MCR:SATB2 primary tumor transplant into transparent Casper zebrafish, 3.5 weeks post-transplant.

Article Snippet: Lentiviral particles were produced by co-transfection of 293 T cells with sequence verified pINDUCER20-SATB2, pLentiCMV_SATB2_Blast, or pTK93_Lifeact (Addgene plasmid #46357 was a gift from Iain Cheeseman), and packaging plasmids pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260, both gifts from Didier Trono), using FuGENE HD (Promega).

Techniques:

Journal: Nature Communications

Article Title: Redox regulation of EGFR steers migration of hypoxic mammary cells towards oxygen

doi: 10.1038/s41467-018-06988-3

Figure Lengend Snippet: Epithelial cells exposed to deep hypoxia undertake directional migration towards oxygen. a Schematic diagram of the cell confinement assay. b Z -axis confocal analysis of LifeAct-mCherry-expressing MCF10A cells under confinement (+C) or not (NC), showing a layer of ∼7–10 µm of culture medium supplemented with Oregon Green (green) which separates the cells (red) from the coverslip (black). Right panels are magnified regions from left panels. Scale bars, 100 µm (left), 10 µm (right). c Detection of hypoxia under confinement with the Visisens camera demonstrating the formation of a steep oxygen gradient at the edge of the cell cluster. Oxygen level is shown in pseudocolors (also see Supplementary Movie ). d Immunoblot showing HIF1A stabilisation after confinement (24 h). Molecular weights (Mw) are indicated in kDa. e Hypoxia gradient at the edge of the cluster revealed by HIF1A stabilisation as soon as 1 h after confinement. HIF1A levels are represented in pseudocolors. Scale bar, 200 µm. f Representative sequential images of H2B-GFP-expressing MCF10A cells confined (+C) or not confined (NC) showing outwards migration of the cells from the edge of the cluster. Scale bars, 1 mm. g Individual tracking of cells ( n = 24) at the margin of the cell cluster for 48 h, from experiments depicted in ( f ). Tracking is representative of more than three different experiments (also see Supplementary Movies – ). h Directed migration and redistribution of H2B-GFP-expressing MCF10A cells visualised after 48 h of confinement or unconfined. Left panels: bright field images at 48 h with a red dotted-line indicating the front of the cell cluster at 0 h. Middle panels: tracks of H2B-GFP expressing MCF10A confined or unconfined for 48 h. Right panels: relative distribution of MCF10A cells at the edge of the cluster at 0, 24 and 48 h. Scale bar, 500 µm. i Mean values of directionality and speed calculated from ( g ) (mean ± SD; n = 3 independent experiments). See Methods for further information about the calculation of motility parameters. *** P < 0.001, * P < 0.05 by two-tailed Student’s t -test. NC not confined, +C confined

Article Snippet: The lentivector expressing Lifeact-mCherry was constructed by cloning the Lifeact sequence from Addgene plasmid # 54491 into the CSII-EF-mCherry vector.

Techniques: Migration, Expressing, Western Blot, Two Tailed Test