We selected the HepG2, SNU368,

and SNU449 cells as they were found to express little or no HDAC6 by northern and western blot analysis (Fig. 1E), and transfected with pcDNA_HDAC6. As expected, ectopic expression of HDAC6 caused growth retardation and elicited increased LC3B-II conversion in these liver cancer cells as compared with control (non- or empty vector-transfected) cells (Fig. 5A-F). In contrast, for PLC/PRF/5 and SNU423 cells that exhibit relatively high expression of HDAC6 among liver cancer cell lines (Fig. 1E), the knockdown of HDAC6 significantly enhanced growth rates of these cell R788 clinical trial lines (Supporting Fig. 3). Similarly, when the same experimental approach was applied to newly established HDAC6-overexpressing Hep3B cell lines (Hep3B_HDAC6 Clone #1 and Clone #2), resilencing of HDAC6 also caused an increased growth rate compared to control cells (scramble sequence of siRNA transfectants). Lastly, to investigate whether tumor suppressor activity of HDAC6 is HCC-specific,

we selleck screening library analyzed HDAC6 gene expressions of colon, gastric, and breast cancer patients from the NCBI GEO database. We selected two sets of microarray data for each colon, gastric, or breast cancer, and compared HDAC6 expression in cancer patients with that of nontumor tissues. There were no significant differences of HDAC6 expression between the normal and tumor group in both colon and gastric cancer datasets (Supporting Fig. 5A-D), whereas the HDAC6 expression in breast cancer was variable depending on cohort study (Supporting Fig. 5E,F). However, when ectopic overexpression of HDAC6 was performed in each of three different colon, gastric, or breast cancer cell lines, all cell lines exhibited no changes in growth rate and LC3B-II conversion (Supporting Figs. 6-8). These results clearly indicated 上海皓元 that HDAC6 functions as a tumor

suppressor by activating autophagic cell death, and tumor suppressor activity is specific to HCC. To investigate whether the stable overexpression of HDAC6 suppresses liver tumorigenesis, we established two cell lines stably overexpressing HDAC6 (Hep3B_HDAC6 Clone #1 and Clone #2). The functional HDAC6 expression was confirmed by detecting the hypoacetylated α-tubulin in these cell lines (Fig. 6A). These cells also exhibited lower growth rates than mock-transfected cells (Hep3B_Mock; Fig. 6B). The immunofluorescence analysis revealed the apparent accumulation of LC3B in Hep3B_HDAC6 cells, whereas almost no accumulation of LC3B was observed in Hep3B_Mock cells (Fig. 6C). In addition, when cells were examined ultrastructures by transmission electron microscopy, ≈40%-45% of Hep3B_HDAC6 cells exhibited autophagic vacuoles, some of which accumulated to form larger cytoplasmic vacuoles (Fig. 6D-b), and at higher magnifications most vacuoles were found to contain electron-dense material and degraded organelles (Fig. 6D-c,d).