Lsm170

Cells were plated overnight on a 4 or 8-well chamber slide (Millipore or LAB-TEK) coated with poly-L-lysine (Sigma), fixed with 4% paraformaldehyde and washed with PBS. Fixed cells were then permeabilized with 0.5% Triton X-100 in PBS, blocked with 5% BSA, and stained with mouse anti-Tubulin antibody (NOVUS; 1:200 dilution) or human anti-Centromere antibody (Antibody Inc; 1:200 dilution) for 1 h, and incubated with secondary antibodies conjugated with Alexa Fluor dyes (1:500 dilution) for 1 h. Coverslips were then mounted with anti-fade agent Prolong with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Photomicrographs were taken with the NIKON 90i microscope or with the confocal Zeiss LSM170. Analysis and quantification of the mitotic events of interest were performed with ImageJ software (NIH).

H2B-RFP-expressing A549 cells were infected with either shSETD1A or shGFP and real-time imaging was initiated 24 hours after infection using the confocal Zeiss LSM170 using an enclosed stage incubator, after regulating temperature, CO₂ level and humidity. For RFP images within each well, nine images (3 × 3 tile) were captured every 5–8 min over 3 days (24–96 h after infection) at ×20 magnification. Sample focus was maintained during the course of the experiment using an external diode laser. Cell viability was confirmed by observing of mitotic cells throughout the duration of the experiment. Videos were analyzed using the ZEN software (Zeiss).

Confocal images or z‐stacks of protein expression in P. patens protonemata or Arabidopsis roots were captured with the zen 2012 software using the Zeiss LSM170 confocal microscope (20× objective). Wild‐type protonemata and protonemata with uninduced GFP expression were compared with protonemata with induced PpDELLA‐GFP protein expression using identical laser power, gain and pinhole settings. For Arabidopsis, Ler roots were compared to pRGA::GFP‐AtRGA roots. Excitation and emission wavelengths for GFP fluorescence were 488 nm and 530 nm respectively and for chloroplast autofluorescence 634 nm and 696 nm respectively.