Fig 1.
Endoplasmic reticulum (ER) localization of Dpy19L1 in COS-7 cells.
COS-7 cells were transfected with a Dpy19L1-GFP plasmid. After 24 h, the subcellular localization of Dpy19L1 was observed by immunofluorescence for GFP and Calreticulin, a marker for the ER. (A) Confocal images of Dpy19L1-GFP (green) and Calreticulin (magenta). Right panel is the merged image. Dpy19L1-GFP shows a similar pattern with Calreticulin. Arrows indicate localization of Dpy19L1 around the nucleus. (B) Double staining of Dpy19L1 and Calreticulin. Lower image indicates intensity profile analysis along a white line in the upper images. (C) Colocalization analysis. An example of scatter plot of red and green pixel intensities of the Dpy19L1-GFP/Calreticulin double-labeled cell shown in Fig 1A. Intensities were measured from fifteen COS-7 cells. (D) Dynamics of Dpy19L1 and the ER were observed by time-lapse imaging. The movies are shown in S1 and S2 Movies. COS-7 cells were transfected with a Dpy19L1-GFP plasmid together with pER-DsRed2. Upper and lower images show perinuclear (N) and peripheral (P) regions, respectively. Time-lapse images were recorded from twenty-five COS-7 cells. Results shown are representative of at least three independent culture experiments. Scale bars: 20 μm in A and C.
Fig 2.
Distribution patterns of Dpy19L1 along microtubules in COS-7 cells.
(A-C) Double staining of Dpy19L1-GFP (green) and endogenous α-Tubulin (magenta) in COS-7 cells transfected with Dpy19L1-GFP. Nucleus was labeled by DRAQ5 (blue). Lower right panel shows the merged image. Boxed areas are magnified in B and C. Dpy19L1 is highly localized in a perinuclear region (arrows). The meshwork-like pattern of Dpy19L1 along the microtubule network is observed (yellow arrowheads). (D,E) COS-7 cells transfected with a pDpy19L1-GFP plasmid were treated by nocodazole, an inhibitor of microtubule assembly, for 3 h before fixation. (D) Immunostaining of GFP and α-Tubulin. (E) Immunostaining of GFP and Calreticulin. The cytoplasmic reticular staining of Dpy19L1 is severely disrupted by application of nocodazole. Nucleus was labeled by Hoechst 33342 (blue). Results shown here were obtained from at least three independent culture experiments. Scale bars: 20 μm.
Fig 3.
Dpy19L1 distribution in embryonic cortical neurons.
(A) Expression of Dpy19L1 mRNA visualized by ISH on sagittal section of E14.5 mouse embryo. ctx, cerebral cortex; d, diencephalon; p, pons; mo, medulla oblongata; H, heart; Lu, lung; Li, liver; Ki; kidney. (B) Primary neurons were prepared from the E14.5 mouse cerebral cortex and cultured for 5 days. The distribution of Dpy19L1 was observed by immunostaining with anti-Dpy19L1 antibody (C-ter). (C) Double immunostaining for Dpy19L1 (green) and Calreticulin (magenta). Nucleus was labeled by DRAQ5 (blue). Lower right panel shows the merged image. Dpy19L1 is partially colocalized with the ER marker Calreticulin. Arrowheads show Dpy19L1 distribution around the nucleus. C′ and C″ show magnified images of boxed areas. Results shown were obtained from three independent cultures. Scale bars: 200 μm in A, 30 μm in B, and 20 μm in C.
Fig 4.
Distribution of Dpy19L1 along microtubules in embryonic cortical neurons.
(A) Embryonic cortical neurons were double-labeled with anti-Dpy19L1 (green) and anti-α-Tubulin antibodies (magenta). Lower panel shows the merged image. Boxed area is magnified in lower right image. (B) Primary cortical neurons were treated with nocodazole. The cytoplasmic reticular pattern of Dpy19L1 is perturbed by nocodazole treatment. Results shown were obtained from three independent cultures. Scale bars: 10 μm in A and 30 μm in B.
Fig 5.
Requirement of Dpy19L1 in neurite extension of cortical neurons.
(A) COS-7 cells were transfected with either a Dpy19L1 siRNA or control siRNA along with a CAG-Dpy19L1 expression plasmid, followed by western blot analysis at 48 h after transfection. Dpy19L1 expression was efficiently reduced by Dpy19L1 downregulation. α-Tubulin was used as a control. (B) Either control siRNA or one of two Dpy19L1 siRNAs was transfected in embryonic cortical neurons, and 48 h later, the expression level was checked by RT-PCR. Each Dpy19L1 siRNA decreased Dpy19L1 expression. β-actin was used as a control. (A, B) The blots (A) and the gels (B) were cropped from the same gels, and the full-length blots and gels are shown in S5B and S5A Fig, respectively. (C−E) E14.5 cortical neurons were transfected with control or Dpy19L1 siRNA and allowed to differentiate for 72 h. The lengths of the longest neurite were compared between control and Dpy19L1-downregulated neurons. Dpy19L1-downregulated neurons show reduced neurite length compared with that of control neurons. Scale bar: 100 μm. (D) Histogram shows distribution of neurite lengths of control and Dpy19L1-downregulated neurons. (E) Average neurite length of control siRNA and Dpy19L1 siRNA transfected neurons. (F) Comparison of the number of cleaved Caspase3-labeled cells between control and Dpy19L1-downregulated neurons at 48 h after transfection. All data shown here are from at least three independent culture experiments. Values are mean ± SEM. *P < 0.05.
Fig 6.
Distribution of Calreticulin in neurites of Dpy19L1-downregulated neurons.
Either control siRNA (A and C) or Dpy19L1 siRNA1 (B and D) was transfected in embryonic cortical neurons. Cortical neurons were allowed to differentiate for 72 h, subjected to immunocytochemistry for GFP and Calreticulin (A and B), or for GFP and α-Tubulin (C and D). Boxed areas are magnified in A’-D”. Calreticulin, an ER marker, was observed in neurites of both control and Dpy19L1-downregulated neurons. Results shown are representative of at least three independent culture experiments. Scale bars: 50 μm in A-D, 20 μm in A’-D”.