The entire Tween-20 molecule is required to block nuclear tRNA export in HeLa cells.

<p>HeLa cells were treated with 50–300 µM (A) lauric acid (B), SPAN20 and (C) polyethylene glycol for 4 h in serum-free DMEM. The cells were fixed and FISH was used to monitor the location of tRNA^Lys. The cells were stained with DAPI to visualize the nucleus. Scale bar represents 10 µm.</p

Tween-80 or deoxycholate treatment does not affect nuclear tRNA export.

<p>HeLa cells were treated with (A) 150 µM Tween-80 or (B) 150 µM deoxycholate for 4 h in serum-free DMEM. The cells were fixed and FISH was used to monitor the distribution of tRNA^Lys. The cells were stained with DAPI to visualize the nucleus. Scale bar represents 10 µm.</p

Overexpression of NTF2 restores nuclear import of Ran and nuclear export processes in Tween-20 treated HeLa cells.

<p>HeLa cells were transfected with pCMV (Empty) or pCMV-NTF2 (NTF2) for 24 h. The cells were washed and incubated in fresh serum-free DMEM containing 150 µM Tween-20 for 4 h. The cells were then washed and fixed in 1× PBS containing 4% formaldehyde. (A) The cellular location of Ran was monitored by immunofluorescence microscopy or (B) the location of tRNA^Lys was detected by FISH. (C) HeLa cells were co-transfected with NES-EGFP and pCMV or pCMV-NTF2. 24 h post-transfection, the cells were incubated in serum-free DMEM with 150 µM Tween-20 for 4 h and the cellular location of NES-EGFP was monitored by fluorescence microscopy. The nuclei were visualized by DAPI staining. T, transfected cell; NT, non-transfected cell. Scale bars represent 10 µm.</p

Tween-20 treatment causes Ran to accumulate in the cytoplasm of HeLa cells and a block in nuclear export of proteins but not nuclear import of proteins.

<p>(A) Tween-20 causes cytoplasmic retention of Ran in HeLa cells. HeLa cells were incubated in fresh serum-free DMEM (Untreated) or in serum-free DMEM containing 150 µM Tween-20 for 4 h. After the 4 h incubation, the cells were left in Tween-20 containing medium (Tween-20), washed and placed in fresh serum-free media (Wash), or had the serum-free DMEM media containing Tween-20 supplemented with 10% FBS (Serum Add) and incubated at 37°C for 1 h. The distribution of Ran was monitored by immunofluorescence microscopy as described in materials and methods. (B) Tween-20 causes nuclear accumulation of Importin-α. HeLa cells were incubated in serum-free DMEM without (Untreated) or with 150 µM Tween-20 in serum-free DMEM for 4 h (Tween-20). Following the 4 h incubation, the cells were washed and incubated in serum-free DMEM for 1 h. The distribution of Importin-α was monitored by immunofluorescence microscopy. (C) Tween-20 causes nuclear accumulation of an NES-EGFP. HeLa cells were transfected with NES-EGFP and allowed to express for 24 h. Post-transfection cells were washed and placed in fresh serum-free DMEM with (Tween-20) or without (Untreated) 150 µM Tween-20 for 4 h. The distribution of NES-EGFP was monitored by direct fluorescent microscopy. (D) Tween-20 does not block nuclear import of the NLS containing Histone H2A. HeLa cells were incubated in serum-free DMEM without (Untreated) or with 150 µM Tween-20 for 4 h. The distribution of Histone H2A was monitored by immunofluorescence microscopy. (E) Tween-20 does not affect TNF-α stimulated nuclear import of NF-κB. HeLa cells were incubated in serum-free DMEM without (Untreated) or with 10 ng/ml TNF-α for 30 min, or with 150 µM Tween-20 for 4 h and then for 30 min with 10 ng/ml TNF-α. The distribution of NF-κB was monitored by immunofluorescence microscopy. The cells were DAPI stained to visualize the nucleus. Scale bar represents 10 µm.</p

Tween-20 does not affect localization of RanGAP to the NPC.

<p>HeLa cells were incubated in serum-free DMEM (Untreated), serum-free DMEM containing 150 µM Tween-20 for 4 h (Tween-20), serum-free DMEM containing 150 µM Tween-20 for 4 h, washed and then placed in fresh serum-free DMEM for 1 h (Wash), or in serum-free DMEM containing 150 µM Tween-20 for 4 h and supplemented with 10% FBS (Serum add) and allowed to incubate for 1 h at 37°C. The cells were then washed and fixed in 1× PBS containing 4% paraformaldehyde before the distribution of RanGAP (left column) and FG repeat containing nucleoporins (mAb414, middle column) were monitored by immunofluorescence microscopy. Overlay analysis was performed to monitor any change in the localization of RanGAP (right column). Scale bar represents 10 µm.</p

Tween-20 causes an increase in the level of protein phosphorylation at tyrosine and threonine residues, but does not appear to cause modification of NTF2.

<p>(A) HeLa cells were incubated in serum-free DMEM without (Untreated) or with 150 µM Tween-20 (Tween-20) for 4 h. The cells were washed and lysed in the presence of sodium fluoride and sodium orthovanadate (Lanes 1 and 2, -AP) or lysed in the absence of phosphatase inhibitors (Lanes 3 and 4, +AP). The cells lysed without phosphatase inhibitors were incubated with alkaline phosphatase for 30 min at 37°C. Following the incubation, 40 µg of each lysate was separated on 10% gels by SDS-PAGE followed by Western blot analysis to monitor the levels of protein phosphorylation at threonine (first row), tyrosine (second row) and serine (third row) residues or actin level (fourth row). Arrows indicate bands of increased phosphorylation. (B) The lysate (40 µg) prepared as in (A) was separated by SDS-PAGE on a 15% gel followed by Western blot analysis to monitor the mobility of NTF2. The relative mobility of NTF2 was monitored in lysate prepared from Untreated (lanes 1 and 3) and Tween-20 (lanes 2 and 4) treated cells. (C) Lysates treated as in (A) were subjected to chromatography using Phostag™-agarose. The resins were washed and bound proteins were eluted using sodium phosphate containing buffer. Total cell lysate (20 µg) (lanes 1and 3, left) and eluted proteins were subjected to Western blot analysis. NTF2 was detected using α-NTF2 (left) and phospho-Thr proteins (lanes 1 and 2, right) were detected with anti-phospho-Thr antibodies.</p

Prolonged Tween-20 treatment leads to apoptosis.

<p>HeLa cells were incubated in serum-free DMEM (Untreated), serum-free DMEM containing 150 µM Tween-20, or serum-free DMEM containing 25 µM etoposide. The cells were harvested at the time points indicated, lysed and 40 µg of cell lysate was separated by electrophoresis on a 10% SDS-PAGE gel. Western blot analysis was performed to monitor PARP cleavage.</p

Tween-20 does not affect nuclear localization of Xpo-t in HeLa cells.

<p>HeLa cells were incubated in fresh serum-free DMEM (Untreated) without or with 150 µM Tween-20 for 4 h (Tween-20). The cells were washed and fixed with 4% paraformaldehyde in 1× PBS and the distribution of Xpo-t was monitored by immunofluorescence microscopy. The cells were stained with DAPI to visualize the nucleus. Scale bar represents 10 µm.</p

The ERK and PI3K signaling pathways do not control nuclear-cytoplasmic trafficking of NTF2.

<p>(A) HeLa cells were incubated in serum-free DMEM for 4 h (lane 1) and then maintained in serum-free DMEM alone for 4 h, serum-free DMEM containing 150 µM Tween-20 for 4 h (lane 2), or in serum-free DMEM for 4 h and then stimulated with 10% FBS for 30 min (serum) (lane 3). The cells were washed and lysed in the presence of phosphatase inhibitors. 40 µg of each lysate was separated by SDS-PAGE followed by Western blot analysis to monitor the levels of phosphorylated ERK1/2 (p-ERK1/2) (top row) or phosphorylated Akt (p-Akt) (middle row). Analysis of the levels of ERK1/2 (bottom row) was performed as an internal loading control. (B, C and D) HeLa cells were incubated in serum-free DMEM (Untreated), serum-free DMEM containing: 150 µM Tween-20 (Tween-20), 25 µM LY294002 or 50 µM PD98059 for 4 h. The cells were washed and processed for immunofluorescence microscopy to monitor the localization of (B) Ran and (C) NTF2, or for FISH to monitor the localization of tRNA^Lys (D). The nuclei were visualized by DAPI staining. Scale bar represents 10 µm.</p

The nuclear tRNA and mRNA export defect caused by Tween-20 is reversible.

<p>HeLa cells were left in serum-free media (panels A and B, Untreated) or treated with 150 µM Tween-20 for 4 h in serum-free DMEM. The cells were then kept in the presence of Tween-20 (second panel, Tween-20), or washed and placed in fresh serum-free DMEM for 1 h (third panel, Wash). The cells were then fixed and processed for FISH to monitor the distribution of (A) tRNA^Lys or (B) mRNA. (C) Tween-20 treated cells were washed and placed in serum-free DMEM and incubated for the times indicated. The location of tRNA^Lys was monitored by FISH. The cells were DAPI stained to visualize the nucleus. Scale bar represents 10 µm.</p