Titles and legends to supplementary figures
Supplementary Figure 1. Characterization of neuronal-directed differentiation in adult
rat hippocampal NPCs. (A) Schematic representation of neuronal differentiation
procedure. NPCs were plated on poly-ornithine/laminin-coated plates for differentiation
induced by fibroblast growth factor-2 (FGF2) withdrawal and application of retinoic acid
(RA), forskolin and 1% fetal bovine serum (FBS). (B) Immunoblot characterization of
levels of members of the CDK5 signaling pathway and proliferation and lineage
differentiation markers at days 0, 2 and 4 of the differentiation procedure, and qRT-PCR
analysis of levels of CDK5 and p35 at days 0 and 4 of differentiation. Non-transgenic
(nontg) mouse brain homogenate is shown as a positive control for immunoblots showing
markers of mature lineages. (C-E) Live cell imaging of differentiating NPCs at days 0, 2
and 4 of the procedure. (F-H) Immunocytochemical analysis of -III-Tubulin (Tuj1)
immunoreactivity at days 0, 2 and 4 of neural induction. (I-K) Immunocytochemical
analysis of CDK5 immunoreactivity at days 0, 2 and 4 of directed neuronal
differentiation of cultured adult rat hippocampal NPCs. Scale bar = 20 m for low-power
images, 10 m for inset images. * p < 0.05 compared to undifferentiated controls by
unpaired two-tailed Student’s t-test (N = 3).
Supplementary Figure 2. Validation studies of in vitro CDK5 inhibition experiments
with a pharmacological inhibitor (Roscovitine) and siRNA-mediated knockdown
(siCDK5). For experiments with Roscovitine (panels A and B), adult rat hippocampal
NPCs were cultured under neuronal differentiation conditions, and treated with
Roscovitine (Rosco) starting at day 2. For experiments with siCDK5 treatment (panels C-
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H), adult rat hippocampal NPCs were cultured under neuronal differentiation conditions,
and transfected with two different siRNAs targeting CDK5 (siCDK5#1, siCDK5#2) or a
non-targeting control siRNA (Ctrl siRNA). (A) Immunoblot analyses of levels of CDK5,
p35, and actin in day 4 (D4) NPC-derived neural progeny treated with 1 M or 5 M
Rosco for 48 hrs. Brain homogenate from a nontg mouse is shown as a comparative
positive control. (B) LDH-based cytotoxicity assay showing only mild chnages in LDH
release in NPC-derived neural progeny treated with Rosco for 48 hrs. (C) Immunoblot
analyses of levels of CDK5, p35, and actin in day 4 (D4) NPC-derived neural progeny
transfected with Ctrl siRNA or siCDK5. (D) LDH-based cytotoxicity assay showing only
mild chnages in LDH release in NPC-derived neural progeny 48 hrs after transfection
with siRNAs. (E) Protein and mRNA (measured by qRT-PCR) levels of CDK5 in NPCderived neural progeny 48 hrs after transfection with siRNAs. (F, G) Immunolabeling
analysis of CDK5 immunoreactivity in NPC-derived neural progeny treated with
siCDK5. Cell nuclei were co-stained with DAPI reagent. (H) Reduced levels of CDK5
immunoreactivity in NPC-derived neural progeny 48 hrs after transfection with siRNAs.
* p < 0.05 compared to non-targeting siRNA-treated controls by unpaired two-tailed
Student’s t-test (N = 3).
Supplementary Figure 3. Immunoblot analysis of CDK5 activity, and CDK5, p35,
APP/A, cyclin D1, and CDK2 expression levels in the brains of CDK5+/-/APP tg mice.
(A) Brain homogenates were prepared from nine-month old wild-type control mice,
CDK5 heterozygous deficient (CDK5+/-), APP tg mice, or crosses (CDK5+/-/APP tg),
were analyzed by western blot with antibodies against CDK5, phosphorylated CDK5
2
(Tyr15), p35, APP, A, -III Tubulin (Tuj1), and actin as a loading control. (B) Levels of
CDK5 activity, as measured by pCDK5 (Tyr15) immunoreactivity as a marker of
increased activity. (C, D) Semi-quantitative image analysis of levels of CDK5 and p35
immunoreactivity. (E, F), Semi-quantitative image analysis of levels of APP (6E10
antibody) and A 4 kDa monomer (82E1 antibody) immunoreactivity. (G, H) Semiquantitative image analysis of levels of cell cycle proteins cyclin D1 and CDK2. * p <
0.05 compared to wild-type controls by one-way ANOVA with post-hoc Dunnett’s test. #
p < 0.05 compared to APP tg mice by one-way ANOVA with post-hoc Tukey-Kramer
test. N=4 animals per group.
Supplementary Figure 4. Immunohistochemical analysis of A accumulation in the
brains of CDK5+/-/APP tg mice. Brain sections from nine-month old wild-type control
mice, CDK5 heterozygous deficient (CDK5+/-), APP tg mice, or crosses (CDK5+/-/APP
tg), were processed for immunohistochemical analysis with an antibody against A
(4G8). (A-D) Patterns of A immunoreactivity in the cortex. (E) Image analysis of the
percent area covered by A-immunoreactive plaques in the frontal cortex. (F-I) Patterns
of A immunoreactivity in the hippocampus. (J) Image analysis of the percent area
covered by A-immunoreactive plaques in the hippocampus (dentate gyrus, DG). Scale
bar = 1.5 mm (panels A-D); 40 M (panels F-I).
Supplemetary Figure 5. Immunohistochemical analysis of general neuronal integrity in
the brains of CDK5+/-/APP tg mice. Brain sections from nine-month old wild-type control
mice, CDK5 heterozygous deficient (CDK5+/-), APP tg mice, or crosses (CDK5+/-/APP
3
tg), were processed for immunohistochemical analysis with an antibody against NeuN.
(A-D) Patterns of NeuN immunoreactivity in the cortex. (E) Image analysis of the
average number of NeuN-positive cells in the frontal cortex. (F-I) Patterns of NeuN
immunoreactivity in the hippocampus. (J) Image analysis of the average number of
NeuN-positive cells in the hippocampus (dentate gyrus, DG). Scale bar = 1.5 mm (panels
A-D); 100 m (panels F-I). * p < 0.05 compared to wild-type controls by one-way
ANOVA with post-hoc Dunnett’s test. N=4 animals per group.
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