HDAC1 inactivation causes autophagic cell death in Hep3B cells.

<p>(A) Hep3B cells were transfected with HDAC1 siRNA (100 nmol/L) for 72 h, fixed and reviewed under the transmission electron microscope (TEM) (left two panels). With a higher magnification, autophagosomes containing electron dense material and degraded organelles were observed in HDAC1-deficient cells. In contrast, a lot of intact mitochondria were observed in Hep3B cells transfected by si-Scr (right two panels). Arrows indicate the presence of autophagosomes. (B) After knockdown of HDAC1, LC3 conversion (LC3B-I to LC3B-II) was markedly increased, whereas the level of Beclin 1 did not change. Parallel treatment with 5 mM 3-MA for 48 h inhibited LC3B-II activation induced by HDAC1 knockdown. Hep3B cells were also treated with 25 µM ceramide for 24 h as a positive control for the autophagy. The densitometry result of the immunoblots was shown as a bar graph (mean ± SD). Relative protein expressions of HDAC1, LC3B-II and Beclin 1 were normalized with the expression level of control (si-Scr) (*p<0.05). (C) In the immunofluorescence analysis, the cytosolic expression of LC3B was induced by HDAC1 knockdown, and which was reversed by the treatment with 3-MA in Hep3B cells. (D) MTT analysis shows that cell viability was regressed by HDAC1 knockdown in Hep3B cell. The number of viable cells was increased in the parallel treatment of 3-MA for 48 h (*p<0.05). (E) Co-transfection of siRNA targeting LC3B and/or HDAC1. The LC3B conversion was induced by si-HDAC1 in the absence of si-LC3B. The level of proteins were quantified by ImageJ and normalized to GAPDH. Bar graph shows the relative ratio to si-Con transfectant (*p<0.05). (F) MTT analysis shows that the cells viability was recovered apparently in the presence of si-LC3B in HDAC1 knockdown Hep3B cells (*p<0.05, si-HDAC1+si-LC3B vs si-HDAC1).