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Figure S1. Localization of CtBP1 and time course of its neuronal activitycontrolled nuclear shuttling
A. Localization of endogenous CtBP1 in synapses and nuclei in primary neuronal
cultures. Neurons were counterstained for Bassoon or Homer as pre- and
postsynaptic markers; nuclei were stained with DAPI.
B. Cortical neurons with basal activity (control) and at various time-points upon
pharmacological treatment to enhance neuronal activity.
C. Cortical neurons with basal activity or upon block of neuronal activity.
D. LB effect on CtBP1 nuclear shuttling under basal activity of the culture.
E-G. Plots show quantification of the treatments shown in A-C on nuclear IF of
endogenous CtBP1.
Data information: In all graphs columns show means, whiskers indicate SEM, the
numbers in the brackets indicate the number of quantified cells from at least two
neuronal cultures; one-way ANOVA with Bonferroni posttest was used for statistics:
ns– p>0.05,***– p<0.001.Scale bars are 10 µm on the overview image in A, B, C and
D and 5 µm on the close up image in (A).
Figure S2. Long-term imaging does not result in artificial rise in nuclear FPAEGFP
A. Images show no spontaneous PA of CtBP1intPAEGFP during 3h of imaging
before PA with 405nm laser pulse. The scale indicates false color coding of F PAEGFP
intensity. Scale bar is 10 µm.
B. Quantification of the fluorescence changes of CtBP1intPAEGFP in the ROI
depicted in (A).
Figure S3. CtBP1 controls expression of activity-regulated genes in neurons
A. Structure of BDNFpI+II EGFP reporter: promoters I and II (black lines), exons I
and II (boxes), cAMP response element (CRE) and RE1 are depicted.
B. Nuclear IF intensities of CtBP1 are plotted against BDNFpI+II-driven expression
of EGFP for individual cells and show negative correlation.
C-D. Expression of BDNFpI+IIEGFP reporter in primary cortical neurons with basal
vs. enhanced (C) or silenced (D) activity. Cells were counterstained with CtBP1
antibody and DAPI.
E, F. Quantification of the nuclear FEGFP in experiments shown in C and D.
G. Cultured cortical neurons were over-infected with lentiviruses expressing shRNAs
467, 944 or scrambled on the day of plating and harvested at DIV 16. Expression of
synapsin, synaptophysin and CtBP1 was quantified in cell lysates, GAPDH was
always used as a loading control. Synapsin 1,2 and synaptophysin were probed on
the same membrane.
H. Quantification of the effects shown in G. Expression of shRNAs 467 and 944, but
not of scrambled shRNA caused a substantial reduction in the expression levels of
CtBP1 (467: 4±1.2% of control; 944: 2.5±0.7% of control), while having no significant
effect on the expression levels of two other neuronal proteins tested.
I. mRNA expression of 14 activity-regulated genes is regulated after expression of
shRNA467. Normalized mRNA levels relative to levels in neurons expressing
scrambled shRNA are plotted. shRNA 467 yields comparable results as independent
shRNA 944 (Fig 5A), what argues strongly against off-target effects of the shRNAs.
Data information: In the graphs the numbers in the brackets indicate the number of
analyzed cells (E, F) and loadings (H) from two independent experiments or qPCR
reactions derived from samples from three independent cultures (I). Student’s t-test
(E, F and I) or one-way ANOVA with Bonferroni posttest (H) was used for statistics:
ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001. Scale bars are 10µm in C and D.
Figure S4. CtBP1 is slightly reduced at synapses from Bsn-/- mice and is
further depleted in Bsn-/-/Pclo KD neurons.
A, B, C. Synaptic CtBP1 was stained in cultures in which equal numbers of neurons
from WT and Bsn-/- neurons were mixed and plated (A) or in cultures infected with
lentivirus expressing shRNA Pclo28 (B) or scrambled shRNA (C) together with
EGFP-synapsin. Synapses were marked with staining for synaptophysin (A) or
Homer (B,C). In A, synapses from Bsn-/- neurons lack Bassoon specific staining
(arrowhead), which is clearly visible in synapses of WT neurons (arrows). In B and C
synapses of transduced neurons express EGFP-synapsin (arrowheads). Synapses
of neurons expressing Pclo28 shRNA show clear reduction in IF for CtBP1
(arrowhead in B) in comparison to synapses of non-transduced neurons (arrows in
B). Synapses of neurons expressing scrambled shRNA (arrowheads in C) show no
difference in the IF for CtBP1 when compared to synapses of non-transduced
neurons (arrows in C).
D, E. Quantification of synaptic CtBP1 IF in the experiment shown in A, B and C .
F. Immunoblots of expression levels of CtBP1 in total brain lysates of newborn WT,
Bsn-/-, Pclo3 and DKO mutant mice.
G. Quantifications of CtBP1 signal ID in brain lysates from WT, Bsn -/-, Pclo3 and
DKO mutant mice.
H. Images showing the decrease of synaptic CtBP1 IF at the synapses of Bsn -/-/Pclo
KD (Pclo 28) neurons. Synapses were marked by staining for synapsin.
I. Quantification of the synaptic CtBP1 IF in Bsn-/-/Pclo28 neurons.
Data information: Numbers in the brackets on the graphs represent the number of
quantified synapses (D,E) or visual fields (I) from two neuronal cultures and
independent loadings (G). Student’s t-test (D , E and I) or one-way ANOVA with
Bonferroni posttest (G) were used for statistics; ns p>0.05, *** p<0.001. For all
images: Colored images show overlay, color code is given in description, scale bars
are 5µm.
Figure S5. 4AP bicu enhances the neuronal network activity in neurons
deficient for Bassoon and Piccolo
A. Syt1 Ab uptake was used to evaluate the neuronal activity in WT/scrambled and
Bsn-/-/Pclo28 neurons upon 4AP bicu stimulation. Cells were stained after fixation for
synapsin to label synapses. Colored images show overlay.
B. Quantification of the effect shown in A.
Data information: Numbers in the brackets in the graph represent the number of
quantified visual fields from three independent neuronal cultures. One-way ANOVA
with Bonferroni posttest was used for statistics; ns p>0.05, * p<0.05, ** p<0.01, ***
p<0.001. Scale bar is 5µm.
Figure S6. 2DG does not modulate the nuclear and the synaptic CtBP1levels
upon activity block
A. Overexpressed CtBP1intEGFP NAD/H bidning mutant displays aberrant synaptic
localization. Cells were stained with Homer antibody to mark synapses.
B. Enhanced neuronal activity reduces NAD/NADH ratio. Quantification of
NAD/NADH ratio in primary cortical cultures with elevated synaptic activity (4AP
bicu-2.5mM 50µM, 8h) and upon silencing (APV CNQX-50µM 10µM, 48h). Values
are normalized to NAD/NADH ratio in cultures with basal activity (dashed line).
C, D. Quantification of nuclear and synaptic CtBP1 IF from the experiments shown in
E and F.
E, F. Staining of CtBP1 in nuclei (E) and in synapses (F) of neurons in cultures with
basal or blocked neuronal activity and with or without 2DG treatment. Synaptophysin
staining was used to label synapses, DAPI to stain nuclei.
G. Example images showing the propidium iodide (PI) staining to identify dead cells
in control cultures and cultures treated with 5mM 2DG for 48h. Cells fixed with PFA
were used as a positive control. Nuclei are stained with DAPI. White arrows indicate
PI positive cells.
H. Quantification of the effects shown in F.
Data information: In the graphs, the numbers in the brackets indicate the number of
of analyzed samples (B), cells (C) or visual fields (D, H). B shows data of one of two
independent experiments, which yielded comparable results. B,C and H show pooled
data derived from three independent neuronal cultures. One-way ANOVA with
Bonferroni posttest was used for statistics: ns p>0.05, ** p<0.01, *** p<0.001.
Colored images show overlays. Scale bars are 10µm in the overview image in A and
in E, 5µm in the close up in A and in F and 50µm in G.
Supplementary Table S1. Role of CtBP1 and its nuclear shuttling in the
regulation of expression of activity-regulated genes
Table summarizes results of qPCR analysis of expression of activity-regulated genes
in control neurons infected with scrambled and shRNA944. Neurons with basal
activity (control), with enhanced activity and with nuclear exit block were analyzed.
Data information: The data is derived from 6 (scr) or 8 (shRNA944) qPCR reactions
from 2 independent cultures and is normalized to the expression levels of the genes
in neurons with basal activity for each infection. All values are shown as mean ±
SEM. One-way ANOVA with Bonferroni posttest was used for statistics. The full
gene names are listed.