For example, formation of large neurospheres reflects good neurogenic potential of NSCs/NPCs [24]. Therefore, the mouse NSCs/NPCs were treated with different concentrations of prohexadione and trinexapac, and the proliferation of neurospheres were measured. For these studies, the sizes of neurospheres were divided into three different groups: small (<50 μm), medium (50-100 μm), and large (>100 μm). In the DMSO treated control samples 44.93% neurospheres were small, Ibrutinib chemical structure 51.89% were medium, and 3.17% were large in size. Consistent with the results of

our docking and in vitro enzymatic experiments, trinexapac treated NSCs/NPCs did not show any apparent change in the number, morphology, or size of neurospheres ( Figure 2a). However, with an increase in the prohexadione concentration, the size distribution of neurospheres were 53.14% small and 46.85% medium at 1 mM; 74.83% small and 25.16% medium

at 1.5 mM; and 75.81% small and 24.18% medium at 2 mM ( Figures 2b and c). Interestingly, large neurospheres normally seen in neurosphere assays, 3.17% in this case, were completely absent from the prohexadione treated groups, while the numbers of neurospheres in the smaller size range were elevated, indicating an inhibition of neurosphere proliferation ( Figure 2c). Thus, consistent with our docking and biochemical studies, administration of selected PGRs of the acylcyclohexanediones class had different effect on the growth of neural stem/progenitor cells (NSCs/NPCs), as shown in Fig. 2. Trinexapac, which doesn’t block the Jmjd2a second demethylase activity, fails TSA HDAC mw to affect the growth potential of NSCs/NPCs. On the other hand prohexadione, which blocks the Jmjd2a demethylase activity, significantly reduces the growth potential of

NSCs/NPCs in a dose dependent manner ( Fig. 2). Taken together, our results indicate a clear correlation between the inhibition of demethylase activity and the stem cell growth by selected PGRs. Finally, we evaluated if prohexadione-mediated inhibition of neurosphere proliferation is mediated via inhibition of demethylation on H3-K9, H3-K27 and H3-K36 sites by immunofluorescence studies. To this end, no significant change was observed in the methylation status of H3-K9me2 mark (data not shown); however, the H3-K27me2 and H3-K36me2 levels increased with an increase in the concentration of prohexadione ( Figure 3). These studies indicate that prohexadione likely acts in vivo by inhibiting H3-K27 and H3-K36 specific demethylases (e.g. Jmjd3 and Jmjd2a) [25]. Since the dynamic histone lysine methylations, particularly of H3-K27 residue, play critical roles in neural stem cell proliferation, stem-ness and differentiation [21], [22] and [23], we evaluated the cellular fate of prohexadione treated neurospheres by immunofluorescence studies using antibodies for neuronal nuclei or NeuN, a neuronal marker, and for glial fibrillary acidic protein or GFAP, a glial marker.