PMC

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 .

Cells were induced for 24 h with doxycycline (1 µg/mL) followed by fixation and extraction. Nuclei were stained with DAPI (blue). DAPI and GFP signal are overlaid in the merge panel. Arrows denote CBs. Double arrowheads mark nucleolar accumulation of S489D and OFF. Triple arrowheads mark small GFP-coilin T122E foci. Scale bars 10 µm.

(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).

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.

Cell counts were obtained 24 h, 48 h and 72 h after seeding with doxycycline or left untreated. The values for each cell line obtained at 72 h were divided by the value of that line at 24 h. Each line was then normalized to the untreated value for that line. Errors bars denote the percent standard error based on an n of no fewer than 5. There is a significant decrease (p value <0.05) in cell number upon the doxycycline induction of GFP-coilin WT or OFF mutant.

HeLa cells were transfected with GFP-coilin (WT) or GFP-coilin phosphomutants (T122E, ON, S489D, S271/272D or OFF). The proliferation rates were measured by conducting a cell titer blue assay 24 h, 48 h, and 72 h post transfection. The values for each transfectant obtained at 72 h were divided by the value of that line at 24 h. All lines were then normalized to WT. Proliferation rates were significantly reduced (p value<0.005) by the expression of all coilin phosphomutants except OFF. Errors bars denote the percent standard error based on an n of no fewer than 6.

Stable cells untreated or induced to express the various GFP-tagged coilin proteins by doxycycline treatment (+) were transfected with control (control KD) or coilin (coilin KD) siRNA. Cells were seeded into 96-well plates and read 72 h later. This corresponds to 96 h post siRNA transfection. The value obtained for 72 h after seeding was normalized to the value obtained for the untreated control knockdown condition for that cell line. WT clone A2F2 was used for these studies. Errors bars denote the percent standard error based on an n of no fewer than 7. There is a significant difference (p value<0.005) between the coilin KD untreated versus doxycycline treated for all cell lines except S271/272D.

(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.