Fig 2: The association of GSTO2 (Anti-GSTO2, Santa Cruz Y-12) and a-tubulin (Sigma, T6074) throughout the 16 steps of mouse spermiogenesis. Mouse spermatids from testicular extracts were fixed in 4% paraformaldehyde and permeabilized after fixation with Triton-X-100. (A) DAPI alone. (B) Anti-GSTO2 antibody alone. (C) Merge A and B. (D) Anti-tubulin antibody alone. (E) A, B, and D merged. Note in steps 13 and 14 that GSTO2 assembles as part of the PAS (arrows). Blue = DAPI, red = anti-GSTO2 (Y-12), green = anti-tubulin, yellow = the colocalization of GSTO2 and tubulin, asterisk = cytoplasmic lobe, and bar = 5 µm. [A color version of this figure is available in the online version.]

Fig 3: eIF3c and eIF3a control expression of module ii and iii subunits of eIF3 and link all three modules tightly together in HeLa cells. (A) A schematic model of human eIF3 and its binding partners combining findings presented here and elsewhere (7, 10, 13, 14, 26, 46). Individual eIF3 modules as well as eIF3 subunits forming the PCI/MPN octamer are indicated. An arrow indicates a potential interaction between eIF3i and -g as proposed in reference 10. (B) Effect of siRNA targeting eIF3j (2 nM), eIF3c (5 nM), and eIF3a (5 nM) mRNAs on protein levels of eIF3 subunits and other eIFs determined by Western blotting 72 h posttransfection (this experiment was repeated numerous times with similar results). NT, control nontargeted cells; Tub, loading control, anti-TUBA4A from Sigma, catalog no. T6074. WCEs were prepared as described in Materials and Methods. Western blots were quantified using Quantity One and/or NIH ImageJ, and the signals were normalized to a-tubulin (Tub). The resulting values obtained with the control NT cells were set to 1.00, and those obtained with eIF3jKD, eIF3cKD, and eIF3aKD cells were expressed relative to the NT. Standard deviations (SD) from at least three individual experiments are given. (C) Effect of siRNA targeting eIF3j (2 nM), eIF3c (5 nM), and eIF3a (5 nM) mRNAs on integrity of eIF3 determined by the coimmunoprecipitation assay using eIF3b as a co-IP bait (anti-eIF3b antibody from Santa Cruz, catalog number sc-16377) followed by Western blotting 72 h posttransfection (see also Fig. S3 in the supplemental material). nc, negative control (beads only, no antibody). Western blots were quantified using Quantity One and/or NIH ImageJ, and the signals were normalized to eIF3b. The resulting values obtained with the control NT cells were set to 1.00, and those obtained with eIF3jKD, eIF3cKD, and eIF3aKD cells were expressed relative to the NT (SD are given).

Fig 4: Hypoglycosylation levels of the CaV regulatory a2d subunits under different concentrations of tumicamycin. Immunoblot analysis of glycosylated fragments of a2d subunits heterologously expressed in HEK293 cells (along with the a1A CaV2.1 pore-forming channel subunit and ß3 regulatory subunit), treated or not with increasing concentrations of tunicamycin (0.2, 0.6 and 2 µg/mL, as indicated). The extent of a2d glycosylation was identified by using antibody anti-a2 (1:500 dilution, Sigma D219, St. Louis, MO, USA) (top panels) and is shown as the difference in molecular weight between the glycosylated and unglycosylated forms. Molecular weight markers are indicated on the left. PNGase F was also added to protein extraction from tunicamycin-untreated cells in order to identify unglycosylated forms in vitro. Upper bands potentially corresponding to glycosylated a2d progressively decrease as the concentration of tunicamycin increases, which in turn promotes the appearance of a lower band potentially corresponding to unglycosylated a2d (lanes 2 to 6). In vitro PNGase F treatment (lane 1) shows two main bands potentially corresponding to unglycosylated a2d (upper band) and unglycosylated a2 (lower band), as previously reported [26].Protein extraction from HEK293 cells transfected only with the vector plasmid was included as negative control (lane 7). For each experimental condition, the protein sample was probed with anti-tubulin (1:2000 dilution, Sigma T6074) (bottom panel) as loading control.

Fig 5: Upon inhibition, DYRK3 relocalizes to splicing speckles and promotes aberrant SC35 mitotic body accumulation. Related to Fig 3 A–CHeLa cells were left untreated or treated with GSK621666 (1 µM) for 2 h. Cells were fixed and stained for DYRK3 and SC35. Scale bar is 10 µm. (B) Automatic segmentation of nuclear speckles is based on SC35. An automated imaging assay was used to quantify the percentage of nuclear speckles highly enriched for DYRK3 (using a fluorescent ratio 1.5 as threshold). Number of speckles quantified = 5,600 (control); 9,794 (GSK 2 h); P < 10-10 (Student’s t-test). (C) Quantification of the percentage of mitotic cells showing aberrant SC35 mitotic bodies. n = 4 independent experiments, ± s.e.m. Number of cells counted: 719 (control); 720 (GSK 2 h); P = 0.0001 (Student’s t-test).DHeLa cells were left untreated (control) or treated with GA (5 µM), 17AAG (5 µM), or VER (10 µM) for 8 h. Cells were then fixed and immunostained for SC35 and P-Ser10-H3 antibodies. Scale bar is 10 µm.EHeLa cells were left untreated or treated with GSK621666 (1 µM) or GA (5 µM) for 2 h. Cells were fixed and stained for DYRK3 and Hsp90. Scale bar is 10 µm.FHeLa cells overexpressing GFP-DYRK3 were fixed 24 h post-transfection. Nucleoli were visualized with an antibody specific for fibrillarin. Scale bar is 10 µm.GHeLa cells were lipofected with a cDNA encoding for GFP-DYRK3 or GFP-DYRK3-dN. 24 h post-transfection, cells were either left untreated or exposed to GA (5 µM) for 8 h. The subcellular distribution of GFP-DYRK3 and GFP-DYRK3-dN was investigated by confocal microscopy. Splicing speckles were visualized with an antibody specific for SC35. Scale bar is 10 µm.H24 h post-transfection, HeLa cells overexpressing GFP-DYRK3 or GFP-DYRK3-dN were lysed and cell lysates were subjected to immunoprecipitation of GFP proteins. Levels of GFP and endogenous Hsp90 are shown in the input and bead fractions.IReplacing the N-terminus of DYRK3 with the Sup35-NM domain (Sup35-NM-GFP-DYRK3-dN) restores DYRK3 targeting to stress granules. 24 h post-transfection, HeLa cells overexpressing Sup35-NM-GFP-DYRK3-dN were either left untreated or exposed to arsenite (500 µM) for 45 min. Sup35-NM-GFP-DYRK3-dN recruitment inside SGs was investigated by confocal microscopy. SGs were visualized with an antibody specific for G3BP. Scale bar is 10 µm.JConfocal microscopy image of HeLa cells expressing for 24 h Sup35-NM-GFP-DYRK3-dN and either left untreated (control) or exposed to GA (5 µM) for 8 h. DAPI was used to stain nucleic acid. Scale bar is 10 µm.KTotal proteins were extracted from HeLa cells treated as described in (J); GFP and TUBA4A protein levels were analyzed by immunoblotting.