fected SH-SY5Y cells. Apoptotic Index is the percentage of cells displaying apoptotic nuclei after treatment with STS for 12 h. doi:10.1371/journal.pone.0024473.t001 In further support of the notion that Hsp72 is involved in the protection against STS, thermally preconditioned undifferentiated SH-SY5Y and 5YHSP72.1 cells became highly susceptible to apoptosis in the presence of KNK437 and STS. KNK437 is a benzylidene lactam compound reported to inhibit thermotolerance by blocking the upregulation of inducible Hsp72. KNK437 has been found to be more effective at inhibiting the inducible Hsp72 protein compared to the broad spectrum heat shock inhibitor quercetin. It has been shown that KNK437 functions by blocking the induction of Hsp70 at the mRNA level. Therefore, it is possible that KNK437 inhibits other heat shock proteins which involve the activation of heat shock factor 1. This drug has also been reported to inhibit expression of Hsp105 and Hsp40. Further details of how KNK437 functions needs to be investigated in future studies. Nonetheless, we have shown that at non-toxic concentrations, KNK437 effectively restricts inducible Hsp72 expression to control levels in our cell model. In spite of possible off-target effects, the data obtained using KNK437 are consistent with Hsp72 being an important factor in both neuronal heat resistance and protection against apoptosis. NSC34 cells against apoptosis associated with the presence of mutant SOD1 aggregates, yet does not affect the frequency of aggregate formation. An understanding of the interplay between Hsp72 levels and cell survival demonstrated in 
this study, may lead to new strategies for treatment and drug design targeting neuronal survival. Compounds that can up-regulate Hsp72 levels can potentially be used in the treatment of various cerebral insults and neurodegenerative disorders to suppress excessive cell death and allow neuronal function to be retained. Conclusion This study indicates that Hsp72 has a key role in protection against apoptosis and that it acts upstream of Bax activation. The data obtained indicate that the degree of protection to apoptotic stress is dependent not only on the basal levels of Hsp72 but also on the amount “7799399 of Hsp72 induced in the various cellular scenarios analyzed. This study supports the idea that if neurons express beneficial levels of Hsp72 or are exposed to a preconditioning treatment to induce Hsp72, they acquire neuroprotective features that provide these post-mitotic cells with the ability to withstand various stresses, thus enhancing their survival. Roles of Hsp72 in pathological context Hsp72 has been found to be induced in a variety of pathological states, including cerebral ischemia and neurodegenerative diseases, such as Alzheimer’s disease. In experimental mouse models, the expression of Hsp72 is upregulated in the brain several hours after cerebral insult and has been 133407-82-6 correlated with better recovery. In cases of a severe ischemic attack, there is a relatively small induction of Hsp72 in the affected area. Hence, the outcome “7768881 and amount of injury to the area seems to correlate with the expression of Hsp72 detected in the region. Preconditioning has also been used widely in several studies to induce Hsp72 and protect against a subsequent hypoxic insult. In Alzheimer’s disease, where the accumulation of misfolded proteins is the major cause of this neurodegenerative disorder, the increased expression of Hsp72 rescues neurons from the tox