U2OS cells were seeded in a chambered coverglass (Thermo Scientific) and transiently transfected with pcDNA-HOTAIRM1-6× MS2 (full-length, Δ5′, ΔE2 or Δ3′) and pcDNA-MCP-GFP or GapmeRs. Laser microirradiation was performed 48 h post-transfection using a laser-scanning confocal microscope (LSM 780, Carl Zeiss) and a 405 nm laser diode. After laser microirradiation, cells were fixed with 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 in PBS. Immunofluorescence was performed by sequential incubation with primary and secondary antibodies. Nuclei were counterstained in Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Samples were visualized using a laser-scanning confocal microscope (LSM 780, Carl Zeiss) coupled with an image analysis system.
For live cell imaging, U2OS cells that stably expressed GFP-fused Ku70, Ku80 or MDC1 (33 (link)) were transiently transfected with pLKO.1-shHOTAIRM1. Laser microirradiation and time lapse imaging were carried out in a SP5 X inverted confocal microscope (Leica Microsystems) using laser diodes at 405 nm and 488 nm, respectively.
For live cell imaging, U2OS cells that stably expressed GFP-fused Ku70, Ku80 or MDC1 (33 (link)) were transiently transfected with pLKO.1-shHOTAIRM1. Laser microirradiation and time lapse imaging were carried out in a SP5 X inverted confocal microscope (Leica Microsystems) using laser diodes at 405 nm and 488 nm, respectively.
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