Supplemental Figure S1. Validation of Znf179 antibodies. Flag- and HA-tagged mouse
Znf179 proteins were expressed in 293T cells. After 24 h of transfection, total protein
was isolated and subjected to a Western blot analysis with anti-Flag (A), anti-HA (B), or
anti-Znf179 (C) antibodies. Pre-immune serum and antigen-preabsorbed antibody, which
was incubated overnight with the synthetic peptide, were used as control (C).
Supplemental Figure S2. Infection efficiency of the lentiviral shRNA. P19 cells were
infected with the virus (MOI = 4) in 8 μg/ml polybrene and the infection efficiency was
measured by flow cytometric analysis of EGFP expression 48 h after infection.
Supplemental Figure S3. During P19 cell neuronal differentiation, cell viability was
affected by Znf179 knockdown. P19 cells were infected with the control (sh_Neg, against
luciferase) or siZnf179 (sh_2) virus before 2 days of cell aggregation. Cells were
aggregated for 4 days in the presence of retinoic acid (RA) on bacterial-grade dishes then
plated on tissue culture dishes. MTT assays were performed after plating for the indicated
times. Values represent the mean of at least three independent experiments ± SEM.
Statistical analysis was performed using one-way ANOVA with appropriate post hoc tests:
* p<0.05 vs. the non-infected group.
Supplemental Figure S4. Expression patterns of cell cycle-regulatory proteins during
cerebellar granule cell neuronal differentiation. Western blot analysis of indicated protein
expressions from different durations of cerebellar granule cell neuronal differentiation.
DIV, days in vitro.
Supplemental Figure S5. p35 re-expression partially rescues the effect of Znf179
knockdown on P19 cell neuronal differentiation. p35 was inserted into the Znf179
shRNA (sh_2) construct. P19 cells were infected with the indicated viruses before 2 days
of cell aggregation. RT-PCR (A) and Western blot analysis (B) of indicated gene
expressions in virus-infected P19 cells, which were aggregated in the presence of 1 μM
retinoic acid (RA) for 4 days. C, Immunostaining of neuronally differentiated P19 cells,
which were aggregated in the presence of RA for 4 days and then plated in culture dishes
1
for another 6 days, with an anti-MAP2 antibody. D, E, The density and percentage of
MAP2-positive cells was determined in differentiated P19 cells. Cells were counted at
x200 magnification in at least 10 fields of view, and at least 500 cells were counted. The
cells with pycnotic nuclei were not counted. Values represent the mean of at least three
independent experiments ± SEM. Statistical analysis was performed using one-way
ANOVA with appropriate post hoc tests: a p<0.05 vs. the sh_Neg group; b p<0.05 vs. the
sh_2 group.
Supplemental Figure S6. Two transcripts of the mouse Znf179 gene were detected in the
mouse cortex. (A) Schematic structures of human, mouse, and rat Znf179 genes that were
obtained from the Evidence Viewer of NCBI. Taller blocks represent exons and arrows
indicate
the
position
of
primers
5´-ATGGGAAACAGCAGCAACAG-3´;
(forward
reverse
primer:
primer:
5´-GCTGAAGCATCGGATGCAGAAG-3´) used in the PCR reaction. (B) Znf179 gene
fragments were amplified from the indicated cDNA prepared in the presence (+) or
absence (-) of reverse transcriptase. PCR products were analyzed using ethidium
bromide-agarose (2%) electrophoresis.
Supplemental Table S1. Primer list for the gene expression analysis
Supplemental Table S2. Major gene expression changes identified from a microarray of
siZnf179 virus (sh_2 and sh_3)-infected cells vs. control virus (sh_Neg)-infected cells.
Only genes with greater or less than 1.5-fold changes in both sh_2 and sh_3
virus-infected cells are listed.
2