8 well chamber glass slide

TZM-gfp cells (15,000 per well) were seeded in 8-well chamber glass slides (ibidi) in DMEM/10 mendium and allowed to adhere overnight. The following morning the medium was replaced with 200ul of fresh DMEM10 without antibiotics. Purified protein mixtures (300 ng Rev+/− 300 ng Tat) were then transfected using 0.6uL of CrisprMAX reagent (Invitrogen) without Cas9 Plus Reagent, per manufacturer instructions. Cells were incubated 6 h, washed three times with fresh medium, then fed with 200uL fresh DMEM10 medium without antibiotics. Extracellular Tat was then added at 300 ng per well in 200uL total medium and incubated for 24–48 h. Live cells were analyzed by confocal microscopy for induction of GFP reporter signal using a Leica SP5 instrument.

HT1080 cells were seeded on 8-well chamber glass slides (ibidi, Martinsried, Germany), coated with either 10 µg/mL laminin-111 (a gift of Dr. R. Timpl, MPI Martinsried, Germany) or rat-tail collagen I (Gibco), and incubated at 37 °C and 5% CO₂ for 1 h. After washing with PBS, fixation with 4% formaldehyde for 10 min, blocking with blocking buffer (2% horse serum in PBS) and permeabilization with 0.2% triton X-100, cells were stained with a mouse anti-integrin α7 antibody, Clone 3C12 (1:100) in blocking buffer, at 4 °C, overnight. After being washed, cells were incubated with a secondary Alexa 488-conjugated anti-mouse IgG- antibody (1:1000) (Gibco). F-actin and nuclei were stained with Alexa 568-conjugated phalloidin (1:500) (Gibco) and Hoechst dye (1:1000), respectively, for 1 h and 2 min. After mounting with mounting medium (Dako, Hamburg, Germany) images of specimens were taken with a confocal microscope LSM800 with an oil immersion 40x objective (Zeiss, Oberkochen, Germany).

Single-molecule tracking was performed as described in McCann et al. (50 (link)). HEK_293 and HeLa cells were seeded at a density of 20 000 cells/well in 8-well chamber glass slides (Ibidi) coated with 0.5% gelatine 24 h prior to transfection. 200 ng of plasmid DNA/well of either Halo-tagged TDP-43 or mutant TDP-43 was transiently transfected into the cells using X-tremeGENE 9 Transfection Reagent kit (Roche, Basel, Switzerland). HEK-293 cells were incubated at 37°C with 5% CO₂ for 24 h prior to imaging and HeLa cells were incubated at 37°C with 5% CO₂ for 24 h then washed three times and then incubated in DMEM media for another 48 h prior to imaging. 45 min prior to imaging 1nM of JF549 Halo-tag dye was added directly to the media and cells were incubated for 10 min at 37°C with 5% of CO₂. Following incubation, cells were washed twice 15 min apart and replaced with Fluorobrite DMEM (Glibco).