Expression of GFP-coilin and GFP coilin phosphomutant proteins in endogenous coilin depleted stable cell lines.

<p>(A and B) Stable cell lines were transfected with coilin siRNA (+) to deplete endogenous coilin, or control siRNA (−). 24 h post siRNA transfection, GFP-coilin or GFP-coilin phosphomutant expression was induced with doxycycline. 24 h later (48 h post siRNA transfection) cells were harvested and lysates were subjected to SDS-PAGE followed by western transfer and probing with anti-GFP antibodies (upper panel). The blots were then probed with anti-coilin antibodies (middle panel) followed by detection of beta-tubulin with anti-beta-tubulin antibodies (lower panel). KD – and + indicates transfection with control siRNA or coilin siRNA, respectively. Note that the GFP-coilin ON signal was too low to be detected with anti-GFP antibodies and could only be detected with anti-coilin antibodies (A, lanes 5 and 6).</p

Characterization of coilin phosphomutant localization in endogenous coilin reduced stable cell lines.

<p>GFP-coilin and GFP-coilin phosphomutant stable cell lines were transfected with control (Ctrl) or coilin siRNA (KD) for 24 h. 24 h of post siRNA treatment, cells were treated with doxycycline and incubated for another 24 h. The 48 h siRNA transfected and 24 h doxycycline induced stable cell lines were fixed, extracted and immunostained for SMN (red). Nuclei were stained with DAPI (blue). Arrows mark some canonical CBs (containing coilin and SMN). Double arrows mark SMN foci that lack coilin (Gems). Single arrowheads indicate nucleolar coilin accumulation. Double arrowheads mark coilin foci that do not have significant enrichment of SMN, and triple arrowheads indicate dim micro-CB structures. Note that the GFP-coilin ON signal was difficult to detect after the siRNA transfection protocol, so polyclonal GFP antibodies were used to amplify this signal. Scale bars 10 µm.</p

Characterization of doxycycline inducible coilin phosphomutant cell lines.

<p>(A and B) Stable cell lines expressing GFP-coilin of GFP-coilin phosphomutant proteins. Non-induced (−) and 24 h doxycycline induced (1 µg/mL, +) cell lysates of GFP-coilin (WT) and GFP-coilin phosphomutants (T122E, ON, S489D, S271/272D or OFF) were subjected to SDS-PAGE, followed by western transfer. The blots were probed with mouse monoclonal anti-GFP antibodies for specific detection of the GFP-tagged coilin proteins (upper panel). The blots were re-probed with rabbit polyclonal anti-coilin antibodies to detect both endogenous coilin and the GFP-tagged WT and coilin phosphomutants (lower panel). Note that S489D and OFF expression generates an approximately 42 kDa degradation product. (C) Transiently transfected GFP-coilin S489D and GFP-coilin OFF phosphomutant proteins do not have a specific degradation product. HeLa cells were transiently transfected with GFP-coilin S489D and GFP-coilin OFF DNA constructs for 24 h followed by lysate generation, SDS-PAGE and western transfer. The blot was probed with antibodies to GFP. (D) Full length GFP-coilin S489D can be detected after doxycycline induction by immunoprecipitation. Doxycycline (0.33 or 1 µg/mL) induced and non-induced GFP-coilin-S489D stable cell extracts were immunoprecipitated with anti-GFP antibodies. The western blot was probed with anti-GFP antibodies for the detection of the GFP-coilin-S489D fragment (upper panel). The same blot was probed with anti-coilin antibodies for endogenous coilin and full length GFP-coilin-S489D protein detection (lower panel). IgG(H) denotes the immunoglobulin heavy chain.</p

Stable inducible expression of various GFP-coilin proteins in the presence of endogenous coilin.

<p>Expression denotes the ratio GFP-coilin or mutants thereof to endogenous coilin.</p>†<p>Some cells share different combinations of these phenotypes. Therefore, these percentages do not add up to 100. Phenotypes classified as other include SMN foci lacking coilin (Gems), coilin foci lacking SMN, and coilin micro-foci.</p><p>N/A  =  not applicable since the S489D protein is degraded in the stable cell line.</p><p>Proliferation impact in reflects the change in proliferation rate of a given stable cell line with doxycycline compared to the proliferation rate of the same line in the absence of doxycycline.</p

Schematic representation of human coilin showing the locations of the coilin self-interaction domain and apparent nucleolar localization signal (NoLS) [<b>18</b>], nuclear localization signals (NLS), RG box [<b>21</b>] and tudor domain [<b>19</b>].

<p>Also indicated are 11 residues that are phosphorylated. Blue represents phosphorylation sites enriched during mitosis, yellow corresponds to those identified during interphase, and the phosphoresidues identified in both mitosis and interphase are green <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025743#pone.0025743-Toyota1" target="_blank">[9]</a>.</p

Transient expression of various GFP-coilin proteins in the presence of endogenous coilin.

<p>Expression denotes the ratio GFP-coilin or mutants thereof to endogenous coilin.</p><p>The data for the localization of WT, ON and OFF taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025743#pone.0025743-Hearst1" target="_blank">[16]</a>.</p><p>Proliferation impact reflects the change in proliferation rate when compared to the rate of cells expressing WT GFP-coilin.</p

Stable inducible expression of various GFP-coilin proteins with endogenous coilin knockdown.

<p>Expression denotes the ratio GFP-coilin or mutants thereof to endogenous coilin.</p><p>N/A  =  not applicable since the S489D protein is degraded in the stable cell line.</p>†<p>Some cells share different combinations of these phenotypes. Therefore, these percentages do not add up to 100. Phenotypes classified as other include SMN foci lacking coilin (Gems), coilin foci lacking SMN, and coilin micro-foci.</p><p>Proliferation impact reflects the change in proliferation rate of a given stable cell line with endogenous coilin knockdown and doxycycline compared to the proliferation rate of the same line with coilin knockdown but no doxycycline. Rescue indicates that the induced protein increased proliferation in the coilin knockdown background, whereas decrease means the induced protein decreased proliferation rates lower than that found with coilin knockdown alone. No change in proliferation rates in the coilin knockdown background upon induction of mutant is denoted as none (as found for the S271/272 mutant).</p