Fig. 8: Myosin VI anchors RNAPII at transcription sites.

A Cartoon depiction of MVI containing the molecular spring (Flagelliform repeat) inserted proximal to the CBD. At low force, the spring is folded and MVI is in a low actin affinity mode. The application of force leads to extension of the spring which triggers the high affinity actin binding mode. B Example widefield imaging of Halo-MVI (Wild Type) and Halo-MVI-spring stained with JF549 (green) and corresponding Immunofluorescence staining against RNAPII-pSer5 (red) in HeLa cells (Scale bar 5 μm) following MVI knockdown (see Supplementary Fig. 18E for western-blot control for knockdown). Further example images are in Supplementary Figs. 19, 20. C Example STORM render image of MVI (green) and RNAPII-pSer5 (red) following transfection of Halo-MVI and Halo-MVI-spring, as described in the methods (scale bar 2 μm). D Cluster analysis of RNAPII-pSer5 nuclear organisation following treatment in (C). WT refers to Halo-MVI transfection. Individual data points correspond to the average value for a cell ROI. (n = 14 for Normal, 13 for KD, 11 for MVI Spring and 14 for WT) The values represent the mean from the ROIs for each condition (Only statistically significant changes are highlighted *p < 0.05; **p < 0.01; ****p < 0.0001 by two-tailed t test compared to normal conditions). E RT-qPCR Gene expression analysis of 5 serum-responsive genes and housekeeping gene GAPDH with treatment of serum alone, serum following MVI knockdown (MVI KD and transfection with either Halo MVI (WT) or Halo MVI Spring. Data are plotted relative to non-stimulated expression from three independent experiments. Error bars represent SEM from three independent experiments.