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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: J Neurochem. 2015 Feb 8;133(2):174–186. doi: 10.1111/jnc.13029

Figure 1. VPA induces neuronal cell death; inhibited by calpain, JNK and necroptosis inhibitors.

Figure 1

(A) PC12 cells neuronally differentiated as described in MM were treated with VPA (0.05, 0.2, 0.5, 1, 5mM), and assayed for cell death by ethidium homodimer staining 3 and 5 days later. Dead cells (red staining) were counted in 4 quadrants and the % dead cells was calculated and normalized to untreated cells. The results are expressed as % death cells ± SE. (B) PC12, PC70, and PC47 cells were differentiated as in (A). They were treated with VPA (1mM) and assayed for cell death by trypan blue exclusion 1, 2, 3, and 5 days later. Dead cells (blue staining) were counted in 4 quadrants and the % dead cells was calculated and normalized to untreated cells. The results are expressed as % trypan blue+ cells ± SE. (C) Primary rat cortex neurons were mock or VPA (1mM)-treated and assayed for cell death by trypan blue staining 3 days post-treatment. Dead and live cells were counted as in (B) and the % dead cells was calculated and normalized to untreated cells. (D) PC12 cells cultured and differentiated as in (A) were treated with VPA (1mM) alone or with z-VAD-fmk (50 µM), PD150606 (PD, 100 µM), SP600125 (SP, 100 µM) or Nec-1 (Nec, 50 µM) and assayed for cell death by trypan blue exclusion at 1,2,3,and 5 days post-treatment. Dead and live cells were counted as in (B) and the % dead cells was calculated and normalized to untreated cells. The results are expressed as % inhibition ± SE calculated from the formula: 100-((% death with inhibitors/% death without inhibitors) ×100). Maximal inhibitory levels were reached on day 3 post-treatment as determined by one-way ANOVA. (E) Primary rat cortex neuronal cells were treated with VPA (1mM) alone or in the presence of z-VAD-fmk (50 µM) or PD150606 (PD, 100 µM) and assayed for cell death by trypan blue 3 days post-treatment. Dead and live cells were counted as in (B) and the % dead cells was calculated and normalized to untreated cells. The results are expressed as % inhibition ± SE, calculated as in (D). (*P<0.01; **P<0.001)