Journal: Journal of cell science

Article Title: APLP2 regulates neuronal stem cell differentiation during cortical development.

doi: 10.1242/jcs.122440

Figure Lengend Snippet: Fig. 1. APLP2 downregulation changes the positioning of cells in dko cortices. (A) Left: Western blot showing protein levels of APLP2-V5 and actin (loading control) of HEK cells expressing APLP2-V5 transfected with U6 driven APLP2 shRNA1, APLP2 shRNA2 or control shRNA. Right: downregulation of APLP2 normalized to the total actin (n54). (B) Confocal images of coronal slices of wild-type (WT) brains electroporated with constructs expressing APLP2 shRNA or control shRNA together with EGFP, 4 days after electroporation (E14.5–E18.5). APLP2 shRNA expression does not lead to developmental differences in respect to wild-type conditions. (C) Quantification of EGFP-positive cells shown in B. Bar graphs represent frequency distribution of EGFP-positive cells in ten equally divided bins from ventricle (1) to the pial surface (10) of the cortical wall. Values represent the mean 6 s.d. (n55; Student’s t-test). The inset scatter plot compares the population distribution of EGFP-positive cells (n53, only 300–400 cells only are shown for clarity of graph; values represent the median 6 interquartile range; Mann–Whitney test). (D) Confocal images of APP/APLP1 dko cortices transfected with APLP2 shRNA or control shRNA construct coexpressing EGFP, 4 days after electroporation (E14.5–E18.5). APLP2 shRNA expression alters cortical positioning of dko cells (E) Quantification of EGFP-positive cells shown in D. Bar graphs show frequency distribution of EGFP-positive cells in ten equally divided bins from ventricle (1) to the pial surface (10) of cortical wall. Values represent the mean 6 s.d. (n55; Student’s t-test). The inset scatter plot compares the population distribution of EGFP-positive cells (n53, 300–400 cells for clarity of graph; values represent the median 6 interquartile range; Mann–Whitney test). All confocal images are maximal intensity projections of 10–15 consecutive z-sections. Upper cortical layers are stained with Cux1 antibody and DAPI is used for nuclear staining. Scale bars: 50 mm. **P,0.01, ***P,0.001.

Article Snippet: The APLP2 shRNA1 (GI562807) (sequence: 59-CGATTACAATGAGGAGAATCCAACCGAAC-39), the APLP2 shRNA2 (GI562808) (sequence 59-ATGAAGGCTCTGGAATGGCAGAACAAGAC-39) and control shRNA (scrambled sequence: 59-GCACTACCAGAGCTAACTCAGATAGTACT-39) driven by the U6 promoter were obtained from Origene (Rockville, MD).

Techniques: Western Blot, Control, Expressing, Transfection, shRNA, Construct, Electroporation, MANN-WHITNEY, Staining

Journal: Journal of cell science

Article Title: APLP2 regulates neuronal stem cell differentiation during cortical development.

doi: 10.1242/jcs.122440

Figure Lengend Snippet: Fig. 3. Progenitor-specific expression of APLP2 shRNA is sufficient to retain cells in the proliferative zone of developing cortex. (A) Schematic comparison of U6-shRNA and microRNA-based shRNA. (B) Western blot showing protein levels of APLP2-V5 and actin (loading control) of HEK cells expressing APLP2-V5 transfected with APLP2 shRNAmir or control shRNAmir. The downregulation obtained by the microRNA construct was about 90% and comparable to the efficiency of the U6 shRNA1 construct (see Fig. 1A). (C) Co-transfection of Ta-cherry (solid arrowheads) with BLBP-GFP using in utero electroporation (E14.5–E16.5) shows that the two promoters target different cell populations because only a very small fraction of cells coexpress mCherry and EGFP (6.12%; 150 cells, open arrowheads). (D) Confocal images (projection of 10–15 consecutive z-sections) of dko cortical slices transfected with APLP2 shRNAmir or control shRNAmir under the control of Ta promoter. Neuronal downregulation of APLP2 does not change cortical positioning. (E) Quantification of EGFP-positive cells shown in D. Bar graphs represent frequency distribution of EGFP- positive cells in ten equally divided bins from ventricle (1) to the pial surface (10) of the cortical wall. Values represent the mean 6 s.d. (n53; Student’s t-test). The inset scatter plot compares the population distribution of EGFP-positive cells (n53, 300–400 cells for clarity of graph; values represent the median 6 interquartile range, Mann–Whitney test). (F) Confocal images (projection of 10–15 consecutive z-sections) of dko cortical slices transfected with APLP2 shRNAmir or control shRNAmir under the control of BLBP promoter. Progenitor-specific expression of shRNA leads to retention of cells in the VZ/ SVZ. (G) Quantification of Cherry-positive cells shown in F. Bar graphs represent the frequency distribution of Cherry-positive cells in ten equally divided bins from ventricle (1) to the pial surface (10) of the cortical wall. Values represent the mean 6 s.d. (n53; Student’s t-test). The inset scatter plot compares the population distribution of Cherry-positive cells (n53, 300–400 cells; values represent the median 6 interquartile range, Mann– Whitney test). **P,0.01, ***P,0.001. Scale bars: 50 mm (C); 100 mm (D,F).

Article Snippet: The APLP2 shRNA1 (GI562807) (sequence: 59-CGATTACAATGAGGAGAATCCAACCGAAC-39), the APLP2 shRNA2 (GI562808) (sequence 59-ATGAAGGCTCTGGAATGGCAGAACAAGAC-39) and control shRNA (scrambled sequence: 59-GCACTACCAGAGCTAACTCAGATAGTACT-39) driven by the U6 promoter were obtained from Origene (Rockville, MD).

Techniques: Expressing, shRNA, Comparison, Western Blot, Control, Transfection, Construct, Cotransfection, In Utero, Electroporation, MANN-WHITNEY