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Analysis of Neurite Length and Apoptotic Features in Transfected Neurons under Various Conditions

This study investigates the effects of TSA, dbcAMP, Actinomycin D, and RNA interference on neurite length and apoptotic features in neuron cultures. Supplementary figures illustrate changes in β-III Tubulin, F-actin, and Hoechst staining, showing variations in average neurite length under different treatments. The analysis includes the impact of CBP/p300 and P/CAF knockdown using RNAi on neuronal health, evident through cleaved caspase-3 staining and the percentage of neurons exhibiting apoptotic characteristics. Results support insights into the molecular mechanisms regulating neuronal growth and survival.

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Analysis of Neurite Length and Apoptotic Features in Transfected Neurons under Various Conditions

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  1. PB Ctrl PB Ctrl 4h 8h 24h 17KD H3 AcK9-14 b-actin 43 KD 120 100 80 60 40 20 0 Supplementary figure 1 A. b III Tubulin F actin Hoechst * B. Average neurite length/cell (µm) Ctrl PB C. ** 160 * 140 Average neurite length/cell (µm) 120 100 80 60 ** ** ** 40 20 0 Ctrl TSA dbcAMP Ctrl TSA dbcAMP Actinomycin D

  2. Supplementary figure 2 A. Ctrl TSA Hoechst β III Tubulin B. TSA Ctrl Hoechst β III Tubulin

  3. Ctrl TSA PDL Myelin 17 KD H3 AcK9-14 43 KD b-actin Supplementary figure 3 A. ** 120 ** 100 ** Average neurite length/cell (µm) 80 60 40 20 0 dbcAMP Ctrl TSA PB B. 120 * * 100 * Average neurite length/cell (µm) 80 60 40 20 0 Actinomycin D Flavopiridol C.

  4. GFP P/CAF Hoechst Supplementary figure Supplementary figure 4 A. 120 RNAi CBP RNAi p300 100 RNAi P/CAF 80 RT-PCR- arbitrary units 60 40 20 0 B. GFP-Scr CBP-p300 siRNA GFP-Scr P/CAF shRNA GFP CBP Hoechst

  5. GFP P/CAF Hoechst Supplementary figure 4 C. GFP-Scr+TSA 1ng/ml CBP-p300 siRNA+TSA 1ng/ml GFP-Scr+TSA 1ng/ml P/CAF shRNA+TSA 1ng/ml GFP CBP Hoechst 120 Ctrl 100 CBP/p300 siRNA 80 P/CAF shRNA Pixel intentiy.-arbitrary units 60 ** 40 ** ** ** 20 0 vehicle TSA

  6. Supplementary figure 4 D. GFP-Scr CBP-p300 siRNA P/CAF shRNA GFP H3 AcK9-14 E. GFP-Scr+TSA 1ng/ml CBP-p300 siRNA+TSA 1ng/ml P/CAF shRNA+TSA 1ng/ml Hoechst

  7. Supplementary figure 4 F. 50 40 30 % of transfected neurons with apoptotic features (fragmented or picnotic nuclei) 20 10 0 GFP-scr P/CAF shRNA CBP/p300 siRNA G. 50 40 30 % of transfected neurons with cleavedCapspase 3 staining 20 10 0 GFP-scr P/CAF shRNA CBP/p300 siRNA

  8. Ctrl TSA Supplementary figure 5 A. AcH3 K9-14 p300 Hoechst AcH3 K9-14 CBP Hoechst B. C. D. TSA TSA 80 140 70 120 120 60 ** 100 Average neurite length/cell 100 50 Arbitrary Units 80 80 ** 40 Arbitrary Units 60 60 30 40 40 20 ** 20 20 10 0 0 0 Ctrl TSA High Low High Low AcH3K9-14 AcH3K9-14

  9. aTub1a +1 SCG-10 +1 Chl1 +1 L1cam +1 CAP-23 +1 Lgals1 +1 -5000 -4000 -3000 -2000 -1000 Supplementary figure 6 A. p53 putative binding site Trans. starting site

  10. 4 4 3 3 2 2 1 1 0 0 4 4 3 3 2 2 1 1 0 0 4 4 3 3 2 2 1 1 0 0 Supplementary figure 6 B. Lcam1 a-Tubulin 1a ** * Fold change TSA / vehicle Fold change TSA / vehicle Ctrl 4h 8h 24h Ctrl 4h 8h 24h CAP 23 SCG10 * Fold change TSA / vehicle Fold change TSA / vehicle Ctrl 4h 8h 24h Ctrl 4h 8h 24h Lgals1 Chl1 * * * ** * * Fold change TSA / vehicle Fold change TSA / vehicle Ctrl 4h 8h 24h Ctrl 4h 8h 24h

  11. Supplementary figure 7 A. 50 40 30 % of transfected neurons with apoptotic features (fragmented or pyknotic nuclei) 20 10 0 GFP p53 K-R B. 50 40 30 % of transfected neurons with cleavedactive caspase 3 positive cells 20 10 0 GFP p53 K-R

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