Fig 2: Upregulation of p75NTR and induction of apoptosis by entinostat in neuroblastoma spheroids. (A,D) Light microscopy images of IMR-32 and SH-SY5Y multicell spheroids (mcs) that were incubated for 72 h with either vehicle or 1 µM entinostat. The scatter plots report the values of mcs sizes expressed as percent of control (vehicle). Scale bar = 200 µm. (B,C,E,F) The spheroids were treated as indicated in (A,D) and then analyzed for p75NTR levels and PARP cleavage by Western blot. The values are the mean ± SD of four individual experiments. ** p < 0.01, *** p < 0.001 vs. vehicle by Student’s t-test.

Fig 3: Induction of p75NTR expression in human neuroblastoma cell lines by VPA and entinostat. (A) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. (B) SH-SY5Y and LAN-1 cells were treated as indicated in (A) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. (C) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.

Fig 4: Prenatal cocaine exposure did not alter the expression levels of full-length (145-KDa) and truncated (95-KDa) TrkB, p75NTR, pro-BDNF and BDNF, Akt1 and ERK2, N-Shc and Shc, as well as NR1 and PLC-?1 in both hippocampus and prefrontal cortex.The expression levels of 145- and 95-KDa TrkB (a). P75NTR (b), pro-BDNF and BDNF (c) as well as Akt1 and ERK2(d), Shc and N-Shc (e), NR1 and PLC-?1(f), in 50 µg post-mitochondrial synaptosome-enriched fractions prepared from hippocampi and PFCX of P21 rats exposed to saline or cocaine in utero were compared by Western blotting. The blots were stripped and re-probed with anti-ß-actin to validate equal loading. Densitometric quantification of blots revealed no discernible differences in 145- and 95-KDa TrkB, P75NTR, proBDNF and BDNF, Akt1, ERK2, Shc and N-Shc, NR1 and PLC-?1 expression levels. n = 4. Data are mean ± s.e.m. of the ratio of 145-, 95-KDa TrkB, p75NTR, proBDNF, BDNF, Akt1, ERK2, N-Shc, Shc, NR1 or PLC-?1to ß-actins optical intensities.

Fig 5: Enhancement of cell surface p75NTR expression by VPA and entinostat. (A) The SH-SY5Y cells were incubated for 24 h with either vehicle, 1 mM VPA, or 1 µM entinostat. Thereafter, the cells were treated with the cell impermeant biotinylating agent sulfosuccinimidyl-6-(biotin-amido) hexanoate and the solubilized proteins were isolated by precipitation with streptavidin-conjugated agarose beads. The total cell extract (cell lysate) and precipitated proteins (surface protein) were analyzed for p75NTR by Western blot. The bar graphs report the changes in the cell surface p75NTR levels that were normalized normalized to pan-cadherin (cadherin), a plasma membrane marker. Values are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t-test. (B) SH-SY5Y cells that were grown on glass coverslips were treated for 24 h with either vehicle (a) or 1 µM entinostat (entin) (b,c), fixed, and incubated overnight with ATTO-488-conjugated anti-p75NTR (extracellular) antibody. In (c) the cells were preincubated with a mouse monoclonal unconjugated antibody directed against the extracellular domain of the receptor before the addition of the labeled antibody. The images were analyzed for p75NTR expression (green color) by fluorescence microscopy. The nuclei were stained in blue with 4'-6-diamidino-2phenylindole dihydrochloride (DAPI). Scale bar = 25 µm. The values that are shown in the scatter plot are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t-test.