TIG3 reduces cell cycle progression.

<p><b>A</b> SCC-13 cells grown on coverslips were infected with 10 MOI of EV or TIG3-encoding virus and after 24 h treated with 10 µM BrdU for 2 h and then fixed and stained with anti-BrdU (red) and anti-TIG3 (green). BrdU incorporation is a marker of the synthesis phase of the cell cycle. The number of BrdU positive cells as a percentage of total cell number is presented beneath each panel. The values are mean ± SEM (n = 3) and the values are significantly different as determined by Student's t-test (p<0.001). Bars = 10 µm. <b>B</b> SCC-13 cells were collected for flow cytometry at 24 h post-infection with EV or TIG3-encoding virus. Cells were stained with 50 µg/ml propidium iodide prior to analysis. TIG3 reduces events in G1 and increases subG1 events. <b>C</b> At 24 h post-infection, cells were harvested and extracts prepared for detection of cell cycle regulatory proteins. Molecular weights are indicated to the left of the blot in kDa. <b>D</b> Cells were treated as above and then harvested for detection of p21 encoding mRNA by real time-PCR. A similar result was observed in each of three experiments.</p

TIG3 localizes in the vicinity of pericentrin.

<p><b>A</b> SCC-13 cells were infected with 10 MOI tAd5-EV or tAd5-TIG3 and at 24 h post-infection were fixed and stained with anti-TIG3 (green). Arrows indicate pericentrosomal and arrowheads indicate membrane localization. No TIG3 is detected in empty vector-infected cells. <b>B</b> Cells, infected as above, were fixed and stained with TIG3 (green) and pericentrin (red). The arrows indicate pericentrin staining of the centrosome and n indicates the nuclei. Bars = 10 µm.</p

Pericentrosomal organelle accumulation in TIG3-positive cells.

<p><b>A</b> SCC-13 cells were infected by 10 MOI tAd5-EV or tAd5-TIG3 and after 24 h fixed and stained to detect TIG3 (green) and various organelle markers (red). The markers include GM130 (cis-Golgi), M6PR (mannose-6-phosphate receptor, trans-Golgi and late endosome), rab11 (recycling endosome), calnexin (ER), and CHC (clathrin heavy chain, intracellular transport vesicle). White arrows indicate pericentrosomal localization of TIG3. Nuclei are stained blue. For GM130, M6PR, and rab11, all panels are red/green/blue merged images. The EV and TIG3 pictures of calnexin staining are red/green/blue merged images. The inserts in the TIG3/calnexin picture are single-color images. For the CHC stain, only the red (CHC) image is shown. Bars = 10 µm. <b>B</b> TIG3 impact on subcellular organelle distribution. To measure organelle spread, the microscope was focused on the z-plane displaying the maximal organelle spread and the area covered by the organelle was monitored using ImageJ Software and presented as organelle area in µm². The values are mean ± SEM (n = 30 fields) including measurement of a minimum of thirty cells per treatment group. Paired Student's t-test analysis identifies the means as significantly different (p<0.001).</p

TIG3 impact on organelle marker protein level.

<p>SCC-13 cells were infected by 10 MOI tAd5-EV or tAd5-TIG3 and after 24 h cell lysates were prepared for immunoblot detection of the indicated proteins. TIG3 expression reduces the level of rab11, GM130 and EEA1, but does not alter LAMP1 level. Molecular weights are indicated to the left of the blot in kDa.</p

TIG3 decreases cell survival.

<p>Subconfluent cultures of SCC-13 cells, growing in 3.8 cm² wells, were infected with 10 MOI tAd5-EV or tAd5-TIG3. At 0, 24, 48, and 72 h post-infection, cells were counted and lysates prepared. <b>A</b> TIG3 expression decreases cell number. Values are mean ± SEM, n = 3. Those comparisons marked by an asterisk are significantly different as determined by Student's t-test (p<0.001). <b>B</b> TIG3 is detected by immunoblot in tAd5-TIG3 infected SCC-13 cells. The monomer is visible at 18 kDa and the bracket indicates higher molecular weight crosslinked TIG3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023230#pone.0023230-Sturniolo1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023230#pone.0023230-Sturniolo2" target="_blank">[11]</a>. Molecular weights are indicated to the left of the blot in kDa.</p

TIG3 induces apoptosis.

<p><b>A</b> SCC-13 cells were infected with 10 MOI of EV or TIG3-encoding virus and after 24 h lysates were prepared for detection of apoptosis markers. The bracket indicates high molecular weight crosslinked TIG3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023230#pone.0023230-Sturniolo1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023230#pone.0023230-Sturniolo2" target="_blank">[11]</a>. Molecular weights are indicated to the left of the blot in kDa. <b>B</b> At 24 h post-infection SCC-13 cells were fixed and stained with anti-cleaved PARP (red). TIG3 increases cleaved PARP staining. The percentage of cleaved PARP positive cells is presented in each panel (mean ± SD). The arrows indicate cleaved PARP-positive cells. Similar results were observed in each of three experiments.</p

TIG3 alters microtubule distribution.

<p><b>A</b> SCC-13 cells were infected with 10 MOI tAd5-EV or tAd5-TIG3 and at 24 h post-infection cells were fixed and stained with anti-TIG3 (green stain) and anti-β-tubulin (red stain). TIG3 accumulates at the expected perinuclear location (black arrow). β-tubulin accumulates in an atypical ring at the cell periphery (red arrows). The normal β-tubulin network in the TIG3-negative cell is indicated by a white arrow pointing to the centrosome. Nuclei were Hoechst stained (blue). The bottom panel is identical to the top, except that only the β-tubulin (red) signal is indicated. Bars = 10 µM. The graph shows the number of tAd-EV and tAd5-TIG3 infected cells with centrosome-originated microtubule arrays. Cells were counted in twenty independent microscope fields and a minimum of one-hundred cells were counted per treatment group. The values are mean ± SEM. Paired Student's t-test analysis reveals that the means are significantly different (p<0.001). To assess the impact of TIG3 on tubulin solubility, cells were infected with tAd5-EV or tAd5-TIG3 and after 24 h total extract, soluble and pellet fractions were prepared and electrophoresed followed by immunostaining to detect β-tubulin and β-actin. The presence of the majority of β-actin in the soluble fraction indicates that the fractionation was successful. <b>B</b> TIG3 expression causes actin filament collapse around the nucleus. SCC-13 cells were infected with adenovirus as above and after 24 h stained with anti-β-actin (red stain) and anti-TIG3 (green stain). Nuclei were Hoechst stained (blue). For the TIG3-positive cells, the left panel shows the TIG3 and β-actin signals (red and green), while the right panel shows only the β-actin (red) channel. The black arrow indicates TIG3 accumulation at the centrosome and the red arrow indicates the β-actin microfilament nuclear ring. Bars = 10 µm. The plot shows the number of tAd-EV and tAd5-TIG3 infected cells displaying actin microfilament rings surrounding the nucleus. Cells were counted in eighteen independent microscope fields and a minimum of one-hundred cells were counted per treatment group. The values are mean ± SEM. Paired Student's t-test analysis reveals that the means are significantly different (p<0.001).</p