PMC:6145447 / 37526-40545 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/6145447","sourcedb":"PMC","sourceid":"6145447","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6145447","text":"Nuclear actin-chromobody-tag-GFP imaging and analysis\nFor live cell imaging experiments, U2OS cells were cultured on 35 mm glass bottom microwell dishes at 90% confluency (MatTek, P35GC-1.5–10-C) and transfected with 0.3 μg of nuclear actin-chromobody-tag-GFP expressing vector (Chromotek, acg-n; EMD Millipore, FuGENE 6 transfection reagent; Promega) After six hours, cells were treated with 1 μg/ml NCS for 60 min at 37 °C. Cells were washed twice with PBS before adding media containing DMSO. After 3 hours, images were acquired on an A1RMP confocal microscope using equipment and settings described above. Cells with actin-cb foci were classified using the following criteria: actin-cb foci exhibited movement and frequent turnover, were round in morphology and often appeared to have fuzzy “tails”. Cells with actin-cb rods were classified using the following criteria: actin-cb rods exhibited limited movement, no turnover, were very bright, and were filamentous in morphology. Foci movements were examined by collecting z series at 0.4 μm intervals throughout the entire nucleus every 30 seconds for 10 min. Following initial image acquisition, cells were subsequently treated with 100 μM CK-666. Images of actin-cb foci were subsequently acquired 1 hour post CK-666 treatment. Actin-cb foci were scored as the number of discrete punctate structures appearing per nucleus, as detected by the Icy Spot Detector plug-in. For experiments assessing localization of actin cb foci with RPA, U2OS cells were co-transfected with actin-chromobody-tag-GFP and RPA32-NLS-mCherry (a kind gift of Jiri Lukas) for 16 hr prior to NCS treatment. After 12 hours, foci movements were examined by collecting z series at 0.4 μm intervals throughout the entire nucleus every 5 min for 2.5 h.\nFor experiments in fixed cells, U2OS cells were cultured on 8-well chamber slides (Thermo Fisher Scientific), subjected to transfection and drug treatments as described above, and processed for IHC as described above. Cells were stained with primary antibodies against Rad51 (Santa Cruz: sc-8349, 1/50) and GFP (Abcam: ab13970, 1/100) overnight. Secondary antibodies were Alexa 488 conjugated goat anti-chicken Ig (Invitrogen: A11039, 1/1000) and Alexa 594 conjugated goat anti-mouse Ig (Thermo Fisher Scientific, A-11005, 1/10000). Actin chromobody-expressing cells were selected for analysis by screening for actin-cb foci. Images were acquired on an A1RMP confocal microscope equipped with a 60×/1.49 Apo-TIRF oil-immersion objective lens. Z series at 0.4 μm intervals throughout the entire nucleus were acquired. For co-localization analysis, actin-cb and Rad51 foci were detected in Z using the spot detector plug-in available on Icy Bioimaging Software platform. The co-localization of Rad51 and actin-cb spots was assessed using the Co-localization Studio plug-in, object-based methodology. Here, co-localization was defined as the number of Rad51 spots that directly overlapped with or touched actin-cb foci (within a 0.7 μm radius).","divisions":[{"label":"Title","span":{"begin":0,"end":53}}],"tracks":[]}