Journal: iScience

Article Title: AKT2-mediated nuclear deformation leads to genome instability during epithelial-mesenchymal transition.

doi: 10.1016/j.isci.2023.106992

Figure Lengend Snippet: Figure 1. TGFb induces nuclear deformation, concomitant with defective nuclear lamina (A) Representative time-lapse images of A549 cells expressing GFP-H2B in the presence (+) or absence () of TGFb are shown. The alteration in the nuclear morphology became more evident 15 h after TGFb stimulation. The arrowheads indicate irregular nuclear shapes. Scale bars, 10 mm. (B) A549 cells were treated with (+) or without () TGFb for 72 h and stained for lamin A (green) and DNA (blue). The insets show a normal nucleus and two deformed nuclei classified as crumpling (a) or lobulation (b). Scale bars, 20 mm. (C) A549 cells were treated with TGFb for various times as indicated and stained for lamin A and DNA. The percentage of the cells with a crumpled or lobulated nucleus was measured (n R 339). (D) A549 cells were treated with TGFb for various times as indicated and stained for lamin A and DNA. The nuclear circularity (4p x area/perimeter2) of the cells was determined (n R 100). (E) Various cancer cell lines, as indicated, were treated with TGFb for 72 h and stained for lamin A and DNA. The percentage of the cells with a deformed nucleus was measured (n R 600). (F) A549 cells were treated with or without TGFb for 72 h and stained for lamin A. The line-scan profiles of lamin A at the selected region (as indicated by dashed lines) are shown using Zeiss Zen2 software. Scale bars, 10 mm. The percentage of the cells with apparent nucleoplasmic distribution of lamin A was measured (n R 246). (G) A549 cells were treated with or without TGFb for 72 h and solubilized in 1% NP40 lysis buffer. An equal proportion of cell lysates from the soluble and insoluble fractions was analyzed by immunoblotting with the antibodies as indicated. The

Article Snippet: Plasmid: HA-Smad2 Addgene Cat#11734 Plasmid: FLAG-Smad3 Addgene Cat#11742 Plasmid: HA-AKT2 Addgene Cat#16000 Plasmid: HA-AKT1 Dr. Chi-Ying Huang (National YangMing Chiao Tung University) N/A Plasmid: FLAG-lamin A This paper N/A Plasmid: pMD.G National RNAi Core Facility (Academia Sinica) https://rnai.genmed.sinica.edu.tw/vector.html Plasmid: pCMV-DR8.91 National RNAi Core Facility (Academia Sinica) https://rnai.genmed.sinica.edu.tw/vector.html Plasmid: pLKO-AS1-puro National RNAi Core Facility (Academia Sinica) https://rnai.genmed.sinica.edu.tw/vector.html Software and algorithms ZEN2 software Carl Zeiss N/A Odyssey CLx Imaging System LI-COR Biosciences N/A Image J NIH https://imagej.nih.gov/ij/ EXCEL Microsoft N/A GraphPad Prism GraphPad https://www.graphpad.com/features Photoshop CS6 Adobe N/A Illustrator CS6 Adobe N/A

Techniques: Expressing, Staining, Software, Lysis, Western Blot