Journal: Neurobiology of disease

Article Title: Depdc5 knockout rat: A novel model of mTORopathy.

doi: 10.1016/j.nbd.2016.02.010

Figure Lengend Snippet: Fig. 2. mTORC1 pathway is upregulated in Depdc5−/−embryos. (A) Schema of the Depdc5-mTORC1 signaling pathway. (B) Top panel: Representative picture of living E21.5 embryos treated with rapamycin (Depdc5+/+, n = 14; Depdc5+/−, n = 23; Depdc5−/−, n = 4). Scale bar: 1 cm. Bottom panel: Histogram showing the percentage of living Depdc5−/−embryos treated or not with prenatal rapamycin. (C) Top panel: Western blot of brain lysates (30 μg of protein) of E12.5 littermate embryos with phosphorylated rpS6 on Ser240/244 (pS6) and actin antibodies. Bottom panel: Histogram of densitometry analysis of pS6 normalized with actin from two independent Western blots. (D) Top panel: Western blot of REF lysates (15 μg of protein) derived from littermate embryos with phosphorylated S6K1 on Thr389 (pS6K1), phosphorylated rpS6 on Ser240/244 (pS6) and actin antibodies after amino acid starvation, with or without rapamycin. Bottom panel: histogram of densitometry analysis of pS6K1 and pS6 normalized with actin from above Western blot. (E) REF soma sizes, expressed as mean cytofluorometric forward scatter (FSC), in standard or amino acid starvation conditions from two independent cytofluorometric REF sizes analysis. Number of embryos is indicated in brackets. Error bars represent mean ± SEM. a.u., arbitrary units; n.s., not significant; *P b 0.05; **P b 0.01; ***P b 0.001 (1-way ANOVA and Tukey's multiple comparison post-hoc tests).

Article Snippet: Immunostaining experiments were performed using standard procedure with primary anti-phosphorylated rpS6 Ser240/244 antibody (Cell Signaling, #5364, 1/200) and secondary fluorescent antirabbit Alexa 488 antibody (Invitrogen, 1/1000).

Techniques: Western Blot, Derivative Assay, Comparison

Journal: Neurobiology of disease

Article Title: Depdc5 knockout rat: A novel model of mTORopathy.

doi: 10.1016/j.nbd.2016.02.010

Figure Lengend Snippet: Fig. 3. Depdc5+/−rats develop brain cortical abnormalities. (A to D) Representative brain anatomy of 4-week old littermate rats (Depdc5+/+, n = 3; Depdc5+/−, n = 3). (A) Nissl-stained whole-brain coronal sections showing the organization of cortical layers. Scale bar: 1 mm. (B) Higher magnification of panel A showing the organization of cortical layers, with enlargement of boundaries between layers I–II and V–VI. scale bar: 100 μm. (C) Top panel; Hematoxylin–eosin staining showing dysmorphic neurons (black arrows) and balloon-like cells (white arrows) in cortical layer V. Middle panel; Nissl staining showing dysmorphic neurons (black arrows) in cortical layer V. Scale bar: 25 μm. Bottom panel; quantitative neuronal size analysis from cortical layers IV–V of 4-week old littermate rats (n = 30 cells per animal). Student's t-test was used. (D) Immunohistochemistry of enlarged cells with intense phosphorylated rpS6 on Ser240/244 (pS6) signals in Depdc5+/−rats. Scale bar: 25 μm. (E) Representative brain anatomy of P11 old (Depdc5+/−, n = 3; Depdc5+/−treated with rapamycin, n = 2) rats. Left and middle panel; Nissl staining showing dysmorphic neurons (black arrows) in Depdc5+/−and normal appearing neurons in Depdc5+/−treated with rapamycin in cortical layer V. Scale bar: 20 μm. Right panel; quantitative neuronal size analysis from cortical layers IV–V of P11 rapamycin untreated and treated rats (n = 30 cells per animal). Kruskall–Wallis test was used. Number of cells is indicated in brackets. Error bars indicate ± SEM. n.s., not significant; ***P b 0.001.

Article Snippet: Immunostaining experiments were performed using standard procedure with primary anti-phosphorylated rpS6 Ser240/244 antibody (Cell Signaling, #5364, 1/200) and secondary fluorescent antirabbit Alexa 488 antibody (Invitrogen, 1/1000).

Techniques: Staining, Immunohistochemistry