Supplementary Figure 1. HDAC-Inhibitors and Dbcamp Promote Neurite Outgrowth, Which Is

Supplementary Figure 1. HDAC-inhibitors and dbcAMP promote neurite outgrowth, which is transcription dependent.

(A) Immunofluorescence staining for β-III tubulin and F-actin in CGN following 0.5 µM PB or vehicle (Ctrl) treatment for 24h. Quantification of neurite measurements from three independent experiments (n = 100), including the average neurite length per cell (µm), is represented in the bar graphs. Scale bar: 50 µm. Neurite measurements show a significant enhancement in neurite length following PB 0.5 µM in CGN at 24h. Asterisks: unpaired two-tailed T-test, * p value < 0.05. Error bars represent SD.

(B) Immunoblotting analysis for H3 AcK9-14 in CGN cultured for 24h under treatment with 0,5 µM Phenyl Sodium Butyrate (PB) or vehicle (Ctrl). Shown is a marked increase in the expression of H3 AcK9-14 following PB. -actin was used as a loading control.

(C) Bar graphs show neurite outgrowth measurements after administration of 1 ng/ml TSA or 1mM dbcAMP in CGN (24 hours) in comparison to vehicle (Ctrl) with and without 1µg/µl Actinomycin D. Quantification of neurite measurements from three independent experiments (n: 100 cells each), including the average neurite length per cell in µm is represented in the bar graphs. Neurite measurements show a significant enhancement in neurite length following TSA (1 ng/ml) and dbcAMP (1mM) in CGN at 24h. Actinomycin D (1µg/µl) significantly counteracts the effects of TSA and dbcAMP in terms of neurite outgrowth. Asterisks: unpaired two-tailed T-test, * p value < 0.05, ** p value < 0,01. Error bars represent SD.

Supplementary Figure 2. TSA promotes neurite outgrowth in CGN plated on myelin and CSPGs.

Phalloidin staining for F-actin and Hoechst shows an increase in neurite outgrowth following 1 ng/ml TSA treatment versus vehicle (Ctrl) when plated on either myelin (A) or CSPGs (B). Scale bar 20 µm.

Supplementary Figure 3. TSA, PB and dbcAMP promote neurite outgrowth in CGN plated on myelin and TSA-dependent outgrowth is transcription dependent.

(A) Bar graphs show neurite outgrowth measurements after administration of 1 ng/ml TSA, 0,5 µM PB, or 1mM dbcAMP in CGN plated on myelin (24 hours) in comparison to vehicle (Ctrl). Quantification of neurite measurements from three independent experiments (n: 100 cells each), including the average neurite length per cell in µm is represented in the bar graphs. Neurite measurements show a significant enhancement in neurite length following TSA, PB, and dbcAMP in CGN at 24h. Quantification of neurite outgrowth was performed from three different experiments (n: 100 cells each). Asterisks: unpaired two-tailed T-test, ** p value < 0.01. Error bars represent SD.

(B) Bar graphs show neurite outgrowth measurements after administration of 1 ng/ml TSA, Actinomycin D (1µg/µl) or Flavopiridol (1 µM) in CGN plated on myelin (24 hours) in comparison to vehicle (Ctrl). Quantification of neurite measurements from three independent experiments (n: 100 cells each). Neurite measurements show a significant enhancement in neurite length following TSA (1 ng/ml) in CGN at 24h. Actinomycin D or Flavopiridol significantly counteract the effects of TSA in terms of neurite outgrowth. Quantification of neurite outgrowth was performed from three different experiments (n: 100 cells each). Asterisks: unpaired two-tailed T-test, * p value < 0.05. Error bars represent SD.

Supplementary Figure 4. Gene silencing for CBP-p300 and P/CAF is efficient, non-apoptotic, and inhibits H3 acetylation.

(A) PC-12 cells were transfected with scrambled (control), CBP-p300 siRNA, or P/CAF shRNA for 24h. RT-PCR was performed to assess mRNA expression levels. Agarose gel bands were quantified by densitometry, normalized to -actin, and compared to control (set as 100%, dotted line) as seen in the bar graph (arbitrary units).

(B) CGN were co-transfected with scrambled vector plus GFP (control), CBP-p300 siRNA plus GFP, or P/CAF shRNA plus GFP and plated on PDL for 24h. Cells were immunostained for CBP or P/CAF, and the nuclei were visualized by Hoechst. Arrows show a strong decrease in protein expression in individual neurons following gene silencing. Scale bar 20µm.

(C) CGN were electroporated as in (B) and treated with TSA (1ng/ml) for 24h on PDL. Cells were stained also for CBP or P/CAF and the nuclei were visualized by Hoechst. Arrows show cells with a decrease of protein expression even with TSA (1ng/ml) when these genes are silenced. Scale bar: 20µm. Bar graphs quantify the expression levels of CBP/p300 and P/CAF as measured by pixel intensity (Image J software) after immunofluorescence with antibodies against CBP or P/CAF in control transfected and silenced CGN with and without TSA (triplicate experiments, n: 80 cell each). Asterisks: unpaired two-tailed T-test, ** p value < 0.01. Error bars represent SD.

(D) and (E) CGN were electroporated as in (B) and plated on PDL with (E) or without TSA (1ng/ml) (D) for 24h. Cells were immunostained for acetylated H3 K9-14 and the nuclei were visualized by Hoechst. Arrows show cells with a decrease of histone acetylation (1ng/ml) when these genes are silenced. Scale bar: 20µm.

(F) Bar graphs show the effects of gene silencing of CBP-p300 and P/CAF on cell survival at 24h as a measure of the percentage of cells with fragmented or pyknotic nuclei (three independent experiments, n:80 cells each). Neither the mean of CBP-p300 siRNA samples, nor that of the P/CAF shRNA samples showed a significant difference from the mean of the control samples. Asterisks: unpaired two-tailed T-test, * p value < 0.05. Error bars represent SD.

(G) Bar graphs show the percentage of GFP-positive transfected neurons expressing cleaved Caspase 3 by immunostaining at 24h after gene silencing of CBP-p300 and PCAF (three independent experiments, n: 100 cells each). The average of cleaved Caspase 3-GFP positive cells does not show statistical difference between neither CBP/p300 nor P/CAF gene silencing and control cells. Unpaired two-tailed T-test, * p value < 0.05. Error bars represent SD.

Supplementary Figure 5.

(A) Immunofluorescence staining for AcH3 K9-14 and CBP or p300 in CGN following TSA (1ng/ml) treatment for 24h. Staining shows differential level of H3 K9-14 acetylation in single cells, which correlates with CBP or p300 staining intensity. Cells with a high level of AcH3 K9-14 show a high level of CBP or p300 (yellow arrows), whereas cells with a low level of AcH3 K9-14 show a low level of CBP or p300 (white arrows).

(B) The bar graph shows average densitometry measurements performed in CGN cells treated with vehicle (Ctrl) or TSA (1ng/ml) (three independent experiments, n: 50 cells each). The bar graph shows an increase of intensity of AcH3 K9-14 in cells treated with TSA compared to control. Asterisks: unpaired two-tailed T-test, ** p value < 0.01. Error bars represent SD.

(C) The bar graph shows densitometry measurements between high immunofluorescence level (=/> mean intensity of TSA treated cells) and low intensity level (=/< mean intensity of vehicle treated cells) of AcH3 K9-14 in CGN (see methods section) (three independent experiments, n: 50 cells each). Asterisks: unpaired two-tailed T-test, ** p value < 0.01. Error bars represent SD.

(D) The bar graph shows a significant correlation between neurite length and intensity of AcH3 K9-14 immunostaining. Asterisks: unpaired two-tailed T-test, ** p value < 0.01. Error bars represent SD.

Supplementary Figure 6. TSA modulates the mRNA expression of several pro-axonal outgrowth putative p53 target genes.

(A) A schematic diagram representing the position of the putative p53 binding sites on each of the gene target promoters.

(B) CGN were treated with vehicle or 1 ng/ml TSA and qRT-PCRwas performed at several time points for different pro-neuronal outgrowth genes which contain a putative p53 binding site in their promoter. Bar graphs indicate quantification of fold increase normalized to untreated cells (three independent experiments). Tubulin1a, SCG-10, Chl1, L1cam and Lgals1, with the exception of CAP-23, were found to be significantly increased in expression following TSA treatment at 24 hours, while the expression of Chl1 and Lgals1was also significantly induced at 4 and 8 hours. Asterisks: unpaired two-tailed T-test, ** p value < 0.01. Error bars represent SD.

Supplementary Figure 7. Neurons electroporated with p53 K-R do not display apoptotic features.

(A) and (B) Bar graphs show the percentage of CGN with apoptotic nuclei (A) or with positive cleaved caspase 3 expression (B) after electroporation with p53 K-R. CGN were transfected by electroporation with p53 K-R or GFP, and stained with Hoechst (24 hours) to observe nuclear morphology (fragmented or pyknotic nuclei) (A) or immunostained for cleaved caspase 3 (24 hours) (B). No significant difference was observed between the mean of control samples and the mean of p53 K-R transfected neurons. (triplicate experiments, n: 100 cells each). Asterisks: unpaired two-tailed T-test, * p value < 0.05. Error bars represent SD.