JCS038950 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Immunofluorescence staining of a U2OS cell expressing the double NLS mutant showed greater accumulation of the protein in the cytoplasm than expression of the single NLS mutans NLS1 and NLS2. Staining was carried out using anti-FLAG monoclonal antibody. Scale bar: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. TDP-43 cellular distribution upon transcription inhibition by treatment with actinomycin D. Endogenous or transiently transfected cells were treated with Act D for 3 hours and then fixed 24 hours post transfection. FLAG-tagged hnRNP A1 and hnRNP C1/C2 were used as controls. Transcription inhibition resulted in cytoplasmic accumulation of endogenous TDP-43 as well as FLAG-tagged wt TDP-43. Scale bars: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Visualization of the nucleosomal ladder following chromatin fractionation of TDP-43-depleted cells. (A) Nuclei from control-treated HeLa cells and HeLa cells treated with siRNA targeting TDP-43 were digested with micrococcal nuclease and separated into three fractions (S1, S2, P). Fractions were digested with protease K and analyzed by agarose gel electrophoresis and ethidium bromide staining. (B) Cells were transfected with siRNA targeting TDP-43 and luciferase control siRNA (mock). Downregulation of TDP-43 was detected by western blotting and tubulin was used as loading control.

• Supplemental Figure S4 -

  Fig. S4. Heterokaryon assays to study the shuttling of TDP-43 mutants. Transiently transfected U2OS cells were fused with mouse NIH-3T3 cells upon cycloheximide treatment. Arrows point to mouse nuclei of the fused cell; human nuclei were distinguished from mouse nuclei through DAPI staining. Localization of FLAG-tagged TDP-43 mutants was visualized by immunofluorescence using anti-FLAG monoclonal antibody. Scale bars: 10 µm.