Consistent with overexpression of a H3K36me2-specific demethylase KDM2B in T-cell and B-cell acute lymphoblastic leukemia and in acute myeloid leukemia, we found its overexpression in almost all hematopoietic cell lines

lysosomal translocation of GAPDH-HT would ” occur in HeLa cells with siRNA-mediated knockdown of LAMP2A. We designed a LAMP2A-siRNA that targets an RNA sequence corresponding to the carboxyl terminus of LAMP2A, which is unique to LAMP2A and absent in other LAMP2 isoforms. Transfection with LAMP2A-siRNA nearly abolished the expression of LAMP2A while only partially decreasing the amount of total LAMP2, suggesting that LAMP2A-siRNA specifically targets and decreases LAMP2A among the three LAMP2 isoforms. Compared to nontargeting control siRNA, LAMP2A-siRNA significantly inhibited the dotlike accumulation of GAPDH-HT in transfected cells. Furthermore, CMA is reported to be activated by various stimuli, including long-term serum deprivation, oxidative stress and mycophenolic acid, which decrease intracellular GTP levels. Therefore, we monitored the translocation of GAPDHHT in the presence or absence of these treatments for 21 h following labeling with the TMR-HT ligand. The proportion of cells showing dot-like accumulations of GAPDH-HT was significantly increased by serum deprivation, 100 mM H2O2 and 10 mM MPA. In addition, the number of dots per cell was also increased by these treatments. In contrast, CMA is inhibited by the inhibitors of p38 mitogen activated protein kinase and by cycloheximide. Indeed, lysosomal translocation of GAPDH-HT was significantly inhibited by 20 mM SB202190, a p38 MAPK inhibitor and 20 mg/ml cycloheximide. These results indicate that CMA activity can be purchase GW 501516 assessed by monitoring lysosomal translocation of GAPDH-HT, representing a novel method to evaluate CMA in individual cells. Since macroautophagy is considered a nonselective protein degradation pathway for cytosolic proteins, it is possible that lysosomal accumulation of GAPDH-HT is mainly mediated by macroautophagy, not by CMA. To exclude this possibility, we examined lysosomal translocation of GAPDH-HT in cells treated with siRNA against Atg5, which is essential for macroautophagy. We confirmed that Atg5-siRNA decreased the amount of Atg5Atg12 complex and macroautophagy activity. We also examined the effects of 3-methyladenine, which suppresses macroautophagy by inhibiting type III phosphatidylinositol 3kinase, on translocation of GAPDH-HT. Lysosomal translocation of GAPDH-HT was not affected by Atg5-siRNA or 10 mM 3-MA. Moreover, GAPDH-HT accumulated in LAMP2A-positive lysosomes in embryonic fibroblast cells from Atg5-knockout mice. These findings suggest that the lysosomal accumulation of GAPDH-HT is not mediated by macroautophagy. Macroautophagy is activated to compensate for CMA impairment by LAMP2A knockdown. In the present study, LAMP2A-siRNA increased the dot-like immunostaining of LC3 and the amount of LC3-II, which is specifically localized to autophagosomes , confirming that LAMP2A-siRNA activated macroautophagy in HeLa cells. Interestingly, LAMP2AsiRNA increased the abundance of the Atg5-Atg12 complex detected with the anti-Atg5 antibody, suggesting that CMA affects macroautophagy activity by regulating Atg5-Atg12 complex levels. If GAPDH-HT accumulated in lysosomes by macroautophagy, this accumulation would be stimulated by LAMP2A-siRNA. However, accumulation was inhibited by LAMP2A-siRNA. Moreover, dots of GAPDH-HT rarely colocalized with dot-like immunostaining of LC3 even in the presence of CMA activators, while they strongly colocalized with or were surrounded by LAMP2A immunoreactivity. Collectively, February 2012 | Volume 7 | Issue 2 | e31232