Figure 1.
Nischarin is highly expressed in the brain of adult rats.
(A) Total mRNA extracted from heart, lung, liver, kidney, stomach, small intestine, brain and spinal cord of adult rats was assayed by quantitative real-time PCR (n = 5). Relative quantification was assessed by normalizing the amount of Nischarin to the housekeeping gene GAPDH. (B, C) Protein samples from different tissues were analyzed by Western blot and the quantitative analysis was performed by normalizing the intensities of the hybridization signals to GAPDH (n = 5). Nischarin is highly expressed in liver, brain and spinal cord at both the mRNA and protein levels. Data are presented as mean ± SD.
Figure 2.
Regional distribution of Nischarin in the rat brain.
The highest expression level of Nischarin mRNA was in the cortex, based on real-time PCR (A). This was confirmed by Western blot analysis and immunostaining data at the protein level (B, C, E). Data are presented as mean ± SD. n = 5. The overview immunofluorescence images and the higher magnification images (*) revealed that Nischarin was clearly present in the pyramidal neurons of the cerebral cortex, the CA subfields of the hippocampus, and the Purkinje cells in the cerebellum (D). DG, dentate gyrus; ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer; WM, white matter. Scale bars, 200 μm. Images are representative of 3 rats.
Figure 3.
Subcellular expression pattern of Nischarin in neuronal cell lines.
(A) Western blot data revealed the expression of Nischarin protein in both PC-12 and Neuro-2a cells. MCF-7 cells and cortical tissue lysates were used as the positive controls (n = 5). (B) Immunofluorescent double-staining was performed to reveal the subcellular distribution of Nischarin in PC-12 cells. Co-localization of Nischarin (red) and Map-2 (green) was observed in the cytoplasm and dendrite-like projections (arrowheads in B). (C) The subcellular distribution of Nischarin was further confirmed by staining Neuro-2a cells with Nischarin antibody (red) and FITC-phalloidin (green). Strong staining for Nischarin in the perinuclear region and F-actin-rich protrusions (arrows in C) was observed on the overview and the higher magnification images (*). Scale bars, 5 μm.
Figure 4.
Knockdown of endogenous Nischarin promotes cell migration.
PC-12 and Neuro-2a cells were transfected with anti-Nischarin siRNA or control siRNA. (A) Immunoblot data showed that expression of endogenous Nischarin, but not that of integrin α5 was remarkably reduced at 48 h after transfection in Neuro-2a cells. (B) Cells migrating across the membrane of the transwell were stained with DAPI. Scale bar, 20 μm. (D) Images of migrated cells subjected to scratch assays. Scale bar, 100 μm. The dotted straight lines indicate the dimensions of the scratch, and the solid irregular lines indicate the cell edges. (C, E) Quantitative measurements of the motility indicated enhanced migration in cells transfected with anti-Nischarin siRNA compared with the control siRNA. (F) Proliferation rates of Neuro-2a cells are determined using MTT assay over 48 h. Data are presented as mean ± SD. n = 9/group. One-way ANOVA. *p<0.05, **p<0.01.