Data supporting Genetically encoded lysine photocage for spatiotemporal control of TDP-43 nuclear import

Here, we demonstrate spatiotemporal control over the nuclear import of TDP-43 by installing a photocage (<i>ortho</i>-nitrobenzyl ester) on a single lysine residue (K84) through amber codon suppression in HEK293T cells. Scheme 1:· Schematic overview of the site of photocage addition in TDP-43· Schematic of photocage cleavageFigure 1:· Diagram of proposed mechanism· Cell image of HEK293T cells transfected with pCMV-TDP-43 mRuby· Cell image of HEK293T cells transfected with pCMV-TDP-43 K84ONBK· Associated Hoechst stainsFigure 2:· Tilescan of TDP-43 mRuby expressing cells showing a variety of expression levels· Tilescan of TDP-43 K84ONBK expressing cells showing a variety of expression levels· Timelapse of TDP-43 K84ONBK expressing cells· Western blot using an anti-TDP-43 antibody and associated Ponceau S stain for different expression constructsFigure 3:· Timelapse of K84ONBK expressing cells post light exposure· Data associated with the quantification of nuclear / cytosolic ratio post light exposure as plotted in Figure 3BFigure 4:· Images of cells expressing K84ONBK pre and post exposure with varying intensities of light.· Data used to quantify the final nuclear / cytosolic ratio for each condition as shown in Figure 4B.Figure 5:· Images of cell exhibiting either a nuclear, cytosolic diffuse, cytosolic liquid, or cytosolic solid phenotype that were used for FRAP experiments. Example images of pre-bleaching, time zero, and post-bleaching for each condition are shown.· Data used to quantify the fluorescence recovery after photobleaching shown in Figure 5B.Figure 6:· Cell images of cotransfection with K84ONBK-mRuby and TDP-43-mCerulean. Cells transfected with either plasmid or both together are shown.· Colocalized pixels between the mRuby and mCerulean channels for each condition.Supplementary Figure 1:· Lower magnification view of the cells shown in Figure 1 B and the associated Hoechst stain.Supplementary Figure 2:· TDP-43 K84ONBK mRuby expressing cells co-stained with an antibody for the stress granule marker G3BP1.Supplementary Figure 3:· Cell images taken before and after irradiating a single cell with 355 nm light.Supplementary Figure 4:· Pre and 11 hour post photoconversion performed on a cell with puncta.