Nuclear translocation of the 4-pass transmembrane protein Tspan8

Transmembrane proteins found nuclei reason. To achieve intranuclear localization, the hypothetical transmembrane protein ﬁ rst be extracted from its host membrane and the multiple hydrophobic transmembrane domains must be protected from the hydrophilic environment of the cytoplasm to maintain the proper conformation of the protein. Mechanistically, this poses consider- able challenges.


SiRNA design and transfection
SiRNAs were all ordered from Invitrogen. The siRNA sequence for knocking down 14-3-3θ was CCGACAAGAAGTTGCAGCTGATTAA. The siRNA sequence for knocking down importin-β was CAGTCTGGCTGAAGCTGCTTATGAA. SiRNAs were transfected into MDA-MB-231 cells using a Lipofectamine-3000 transfection kit. The cells were then grown for 48 h for further experiments.

Quick nuclear purification for nuclear protein detection
A 10-cm dish of cells (~1× 10 7 ) were collected and washed in PBS. The cell nucleus was purified by ExKine™ Nuclei Extraction Kit (KTP4002). Nuclear protein samples and total cell lysate samples were separated and detected by western blot assay respectively.
Then the cells were transfected with Tspan8-mCherry plasmid and cultured in the same medium as before for a further 24 h. At the end of the experiment (36 h in total), the cholesterol level of cells was measured by Amplex ® Red cholesterol assay kit (Invitrogen, A12216) according to the manufacturer's instructions.

Nuclear extract preparation
MDA-MB-231 cells expressing Tspan8-mCherry were collected from ten 10-cm culture dishes and washed in PBS. The cell pellet was resuspended in 3 volume of A buffer (10 mM Tris-HCl (pH = 7.9), 1.5 mM MgCl2, 10 mM KCl and freshly added 1 mM DTT) on ice for 10 min with cocktail inhibitors (Roche) and then further lysed by a Dounce tissue grinder (~7 strokes). Next, after 12000 rpm centrifugation for 15 min at 4°C, the suspension and pellet were collected respectively, the suspension as cytosol (stored on ice for further experiments) and the pellet as rough nucleus. Then the pellet was homogenized in 0.5 volume of B buffer (20 mM Tris-HCl (pH = 7.9), 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA (pH = 8) and 20 mM KCl). Then the same volume of C buffer (20 mM Tris-HCl (pH = 7.9), 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA (pH = 8) and 1.2 M KCl) was dropped into the system, and the mixture was gently rotated at 4°C for 30 min. After 10000rpm centrifugation for 30 min at 4°C, the suspension was collected as nuclear extract and the pellet was collected as nuclear pellet. S100 fraction preparation 0.11 volume of 10S100 buffer (300 mM Tris-HCl (pH = 7.9), 30 mM MgCl2 and 1.4 M KCl) were added into the cytosol and the nuclear extract generated during nuclear extrat preparation process, respectively. Then the samples were applied to 100000g ultracentrifugation at 4°C for 1 h. Finally, the S100 and P100 fractions were collected and then for western blot analysis.

Dialysis of nuclear extract
The nuclear extract was dialyzed in 10  volume of D buffer (20 mM Tris-HCl (pH = 7.9), 20% glycerol, 0.2 mM EDTA (pH = 8), 100 mM KCl and freshly added 1 mM DTT) for 4 h. The final product was dialyzed nuclear extract.

Gel-filtration
The chromatographic column (Superose6 Increase 10/300 GL) was pre-equilibrated with more than 30 ml of D buffer. The dialyzed nuclear extract was injected into the column, and separated samples (1 ml each) were collected in tubes. The gel-filtration samples were separated by SDS-PAGE and the proteins were detected by western blot.

Photo-conversion experiments
Photo-conversion experiments were conducted using a NIKON A1 confocal microscope, fitted with a 60× oil objective and 3 times zoom in. The area of interest on the bottom of the cell was photo-converted by 405 nm laser with 10% output for 1 s to partially convert Tspan8-mMaple3 from original green (excited, 488nm laser; emission,~507nm) to red (excited, 561nm laser; emission,~610nm). Immediately after conversion, the focal plane was changed from the bottom of the cell to the nucleus. Then 488 and 561 nm lasers were applied for imaging and each field of 512 × 512 pixels was imaged for a total of 2 h with intervals of 5 min. Finally, the total fluorescent signals in nucleus were traced and analyzed by use of NIS-analysis software.

Diaminobenzidine (DAB) staining and preparation for TEM imaging
MDA-MB-231 cells transfected with Tspan8-mCherry-APEX2 or not (as negative control) were cultured on 3.5-cm dishes overnight. Transfected cells were fixed using room temperature 2.5% glutaraldehyde in APEX reaction buffer (100 mM sodium cacodylate with 2 mM CaCl2, pH 7.4), then quickly moved onto ice for 45 min. Fixed cells were rinsed 5 times in chilled buffer for 2 min, then incubated for 5 min in buffer containing 20 mM glycine to quench unreacted glutaraldehyde, followed by 5× 2 min rinses in chilled buffer. A freshly diluted solution of DAB (1 mg/ml, Sigma) was applied to the cells for 2 min, then 0.003~0.03% (v/v) H2O2 was added into the system at room temperature for 1~15 min depending on the sample. Finally, the cells were rinsed for 5× 2 min in chilled buffer. To prepare the cells for TEM imaging, they were rinsed 3 times in distilled water and incubated in 2% aqueous uranyl acetate (Electron Microscopy Sciences) overnight.
Then the samples were dehydrated in a graded ethanol series (50%, 70%, 80%, 90%, 100%, 100%) for 2 min each on ice, and finally incubated in 100% ethanol for 2 min at room temperature. The cells were then infiltrated and embedded in SPI-Pon 812 resin.
The interesting areas of DAB staining were identified and cut out using a razor blade and mounted on resin blocks with cyanoacrylic adhesive. Finally, trimmed blocks were cut into 70-nm ultrathin sections for examination by TEM (Hitachi, H7650).

Imaging
Confocal microscopy imaging was conducted with an Olympus FV1200 microscope, fitted with a 60× oil objective. Picture size was 1024 × 1024 pixels. Z-stack images were captured using a step size of 0.5 µm from the cell bottom to the cell surface.